U.S. patent application number 13/815042 was filed with the patent office on 2014-02-06 for displaying native human ige neutralizing fcerla-contacting ige b-cell epitopes by constraining super beta(b)-strands and cystine knots on thermostable protein scaffold.
This patent application is currently assigned to Swey-Shen Chen. The applicant listed for this patent is Swy-Shen Chen. Invention is credited to Swy-Shen Chen.
Application Number | 20140039162 13/815042 |
Document ID | / |
Family ID | 50026098 |
Filed Date | 2014-02-06 |
United States Patent
Application |
20140039162 |
Kind Code |
A1 |
Chen; Swy-Shen |
February 6, 2014 |
Displaying native human IgE neutralizing FceRla-contacting IgE
B-cell epitopes by constraining super beta(b)-strands and cystine
knots on thermostable protein scaffold
Abstract
Vaccine displaying native antigenic loops of immunoglobulin E is
critical for eliciting neutralizing anti-IgE antibodies. The
embodiment of the invention enables the display of native antigenic
IgE receptor-contacting loops as IgE B-cell vaccines via three
steps of constraining methods. The loops of multiple antigenic
B-cell epitopes can be molecularly grafted in, and conformationally
constrained by the energy favorable flanking beta (b)-stands, i.e.,
the super b-strands identified in this invention. The constrained
loops can be further stabilized in replacing a selective loop
within the cystine knot peptide. These dual constrained antigenic
loops are then integrated onto thermostable protein scaffolds,
folded in the oxidative milieu that provides further conformational
constraint and high yield.
Inventors: |
Chen; Swy-Shen; (San Diego,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Chen; Swy-Shen |
San Diego |
CA |
US |
|
|
Assignee: |
Chen; Swey-Shen
San Diego
CA
|
Family ID: |
50026098 |
Appl. No.: |
13/815042 |
Filed: |
January 25, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61590778 |
Jan 25, 2012 |
|
|
|
Current U.S.
Class: |
530/387.3 |
Current CPC
Class: |
A61K 2039/6056 20130101;
C07K 2319/00 20130101; A61K 39/0008 20130101; A61K 2039/6068
20130101; A61K 39/00 20130101; C07K 16/00 20130101; C07K 16/4291
20130101 |
Class at
Publication: |
530/387.3 |
International
Class: |
C07K 16/00 20060101
C07K016/00 |
Claims
1. A method of identifying the scaffolding super beta-strands for
the FG loop sequences of the human immunoglobulin E constrained on
a protein scaffold, wherein amino acids from the flanking
N-terminus and C-terminus are truncated, and wherein the FG loop
presented by truncated beta-strands on N- and C-termini is
immunoreactive with neutralizing anti-IgE in non-denaturing
conditions, and wherein the FG loop within the flanking
beta-strands constrained on the protein scaffold is an IgE B-cell
vaccine candidate, whereby neutralizing anti-IgE is elicited in
allergic patients, neutralize circulating and mucosal IgE in
patients.
2. The method of claim 1, wherein the scaffolding super
beta-strands are derived from the sequences the flanking super
beta-strand sequences: YQCRVT and LMPST and the 3' sequence of
TKTSGPR critical for the core FG loop native sequence, HLPR.
3. The methods of claim 1, wherein the loop sequence of the high
affinity IgE receptor binding C2-3 loop, BC, and DE are exchanged
with the native FG loop sequence, and wherein the swapped loop of
C2-3, BC, and DE human IgE sequence, respectively unto the flanking
super beta-strands: YQCRVT and LMPST in addition to the 3' sequence
of TKTSGPR, wherein the swapped C2-3, BC, and DE flanked by the
super beta-stands constrained on the protein scaffold are
immunoreactive under non-denaturing conditions with the
neutralizing anti-IgE, and wherein C2-3, BC, and DE loop swapped
super beta-strands on the protein scaffold are IgE B-cell vaccine
candidates, whereby anti-IgE antibodies elicited by said IgE B-cell
vaccines neutralize circulating and mucosal IgE in allergic
patients.
4. The method of claim 3, wherein the homologous high affinity IgE
receptor-binding sequences, derived from immunoglobulin E of the
feline, canine, bovine, equine, and primate species, are grafted
into the flanking super beta-strand sequences: YQCRVT and LMPST
sequences in addition to the 3' sequence of TKTSGPR constrained on
the protein scaffold.
5. The method of claim 4, wherein the IgE receptor-binding,
species-specific IgE sequences, flanked by super beta-strands on
the protein scaffold are IgE B-cell vaccine candidates of the
respective species, whereby anti-IgE elicited by IgE B-cell
vaccines elicited in the respective species neutralize circulating
and mucosal IgE.
6. The method of claim 1, wherein the protein scaffold belongs to
the green fluorescent protein, and maltose-binding protein.
7. A method of inserting C2-3, BC, DE, and FG loops of human IgE,
flanked by super beta-strands into the loop 5 of EETI-II mutants:
Min-18 with double cystine knots at the terminus of a protein
scaffold, wherein Min 18 is a quadruple derivative of Min-28 with
N-terminal five amino acids deleted, and cysteine 19 mutated into
serine 19, and four amino acids in loop 5, except phenylanaline
deleted, and C-terminus glycine deleted, wherein the C2-3, BC, DE
and FG loops flanked by the super beta-stands inserted in position
of C-FC of loop 5, constrained on the protein scaffold are
immunoreactive under non-denaturing conditions with the
neutralizing anti-IgE, and wherein C2-3, BC, DE and FG loops in
super beta-strands in Min-18 on the protein scaffold are IgE B-cell
vaccine candidates, whereby anti-IgE antibodies elicited by said
IgE B-cell vaccines neutralize circulating and mucosal IgE in
allergic patients.
8. The method of claim 7, wherein the homologous high affinity IgE
receptor-binding sequences, derived from immunoglobulin E of the
feline, canine, bovine, equine, and primate species, are grafted
into the flanking super beta-strand sequences: YQCRVT and LMPST
sequences in addition to the 3' sequence of TKTSGPR inserted in
Min-18 constrained on the protein scaffold.
9. The method of claim 8, wherein the IgE receptor-binding,
species-specific IgE sequences, flanked by super beta-strands on
the protein scaffold are IgE B-cell vaccine candidates of the
respective species, whereby anti-IgE antibodies elicited by IgE
B-cell vaccines elicited in the respective species neutralize
circulating and mucosal IgE.
10. The method of claim 7, wherein the EETI-II mutant is Min-23 of
double cystine knots with N-terminal five amino acids deleted, and
cysteine 19 mutated into serine 19, wherein the C2-3, BC, DE and FG
loops flanked by the super beta-stands inserted in position of P/N
of sequence of CGPNFC of loop 5, constrained on the protein
scaffold are immunoreactive under non-denaturing conditions with
the neutralizing anti-IgE, and wherein C2-3, BC, DE and FG loops in
super beta-strands in Min-23 on the protein scaffold are IgE B-cell
vaccine candidates, whereby anti-IgE antibodies elicited by said
IgE B-cell vaccines neutralize circulating and mucosal IgE in
allergic patients.
11. The method of claim 7, wherein the EETI-II is the natural
occurring Min-28 with triple cystine knots, wherein the C2-3, BC,
DE and FG loops flanked by the super beta-strands inserted in
position of P/N of sequence of CGPNFC of loop 5, constrained on the
protein scaffold are immunoreactive under non-denaturing conditions
with the neutralizing anti-IgE, and wherein C2-3, BC, DE and FG
loops in super beta-strands in Min-28 on the protein scaffold are
IgE B-cell vaccine candidates, whereby anti-IgE antibodies elicited
by said IgE B-cell vaccines neutralize circulating and mucosal IgE
in allergic patients.
12. The method of claim 7, wherein the protein scaffold belongs to
green fluorescent protein, maltose-binding protein, lipocalin.
13. The method of claim 2, wherein the scaffolding super
beta-strands are derived from the sequences the flanking super
beta-strands, YQCRVT and LMPST in addition to the 3' sequence of
TKTSGPR for accommodating neutralizing HIV, influenza, RSV viral
epitopes, recognizing by neutralizing anti-viral antibodies under
non-denaturing conditions.
Description
[0001] This application claims priority in U.S. Provisional
Application No. 61/590,778 filed on Jan. 25, 2012, which is
incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] This invention pertains to novel methods of scaffolding IgE
peptides into native conformation as antigenic vaccine
epitopes.
BACKGROUND OF INVENTION
[0003] Multifactorial pathogenic process of allergic asthma has
posed a challenge in treating this disease. IgE-mediated immediate
hypersensitivity exercises two subsystems in play: (i) Upstream
CD4-T-cells/IgE+ B-cells subsystem: Allergen-specific Th2/Th17 CD4
T-cells contribute to the cytokine-mediated late phase reactions.
In contrast, follicular CD4 (Tfh) T-cells provide essential help to
stimulate IgE+ B-cells in the germinal center (GC) and peri-GC
memory B-cells (Crotty, 2011, Ann. Rev. Immunol., 29: 621). (ii)
Downstream IgE network subsystem: IgE produced by the upstream
Tfh/IgE B-cells initiates and amplifies a complex network of the
inflammatory cell circuit of extraordinary diversity, involving a
web of high affinity IgE receptor (FceRI)-bearing mast cells,
basophils, eosinophils, dendritic cells and Langerhans cells, and
recently neutrophils. Mezzanine intercommunication layer: Histamine
released by mast cells skews dendritic cells (DCs) for Th2
preference (Lambrecht, 2009, Immunity, 31:412). FceRI on mast cells
mediated IgE-dependent antigen presentation, and further augmented
Th2 development (Gong, 2010, BMC Immunol, 2010, 11:34).
[0004] IgE-mediated inflammation can cause the acute phase of
immediate hypersensitivity, and the late phase reaction via a
plethora of IgE-produced mediators; and the IgE-FceRI cellular
network can in turn enhance Th2, and the Th2-mediated late and
chronic phases of allergic asthma. To add further importance to IgE
is the expression of FceRI on airway smooth muscle cells for the
release of TNF-a during intractable asthma. Bronchial epithelial
cells also exhibit FceRI, implicated in released IL-33 and TSLP
that amplify Th2-mediated inflammation (Galli and Tsai, 2012, Nat.
Med., 18:693).
[0005] Besides the IgE/FceRI network, low affinity IgE receptors
(FceRII, CD23) are expressed on nearly all B-cells, which mediate
IgE-dependent antigen presentation for Th2 (Schmaltz, 1996,
Immunol. Invest., 25: 481). FceRII on epithelial cells plays a key
role in retrograde transport of IgE immune complexes in the BAL
fluid, which can therefore play a role in augmenting allergen/IgE
complexes-induced inflammation on intraepithelial mast cells and
airway dendritic cells. The expression of FceRI on IL17AR+
neutrophils strongly suggests a new synergy of IgE and
Th17-mediated inflammation in allergic asthma (Galli and Tsai,
2012, Nat. Med. 18:693; Lambrecht et al., 2009, at. Med., 31:
412).
[0006] Thus, IgE is of paramount importance in the etiology of
allergic asthma by affecting IgE-mediated inflammation, a plethora
of cytokines by multiple cell types, and the profound impact on Th2
(Schmaltz, 1996, Immunol. Invest., 25: 481; Gong et al., 2010, BMC
Immunol., 11:34). Thus a drug candidate such as IgE B-cell vaccine
targeting IgE attenuates inflammation at the multiple levels, in
particular ramification of the IgE-FceRI network. Blunting IgE and
IgE receptors by neutralizing anti-IgE remains the central question
in treating clinical allergic asthma.
[0007] To alleviate or cure the IgE-mediated allergic diseases, it
is imperative to remove circulating and mucosal IgE. In this
regard, the present treatment modality focuses on the removal of
circulating IgE via passive administered monoclonal antibody,
Xolair. Anti-IgE, Xolair that neutralizes the receptor-binding FG
loop of IgE molecules alleviates IgE-mediated allergic asthma
(Chang, 2000, Nat. Biotech., 18:157). In contrast to the passive
monoclonal antibody-based passive vaccine, active IgE vaccines were
proposed as another treatment modality to invoke actively produced
anti-IgE that neutralizes host's IgE. One approach resides in
random chemical coupling of synthetic IgE peptides to the
immunogenic protein carriers as active vaccines (Brown et al.,
2009, U.S. patent application Ser. No. 12/634,336).
[0008] Another embodiment of invention resides in engineering
neutralizing IgE B-cell epitopes within thermostable, immunogenic
protein scaffold in a single step internally (Chen, 2008, U.S.
patent application Ser. No. 12/011,303; Chen, 2008, J. Immunol.
Meth., 333: 10). The present embodiment of the invention represents
constraining native IgE B-cell epitopes in two internal steps: into
super b-strands, and further into the cystine knots; and then
integrated in one external step onto the protein scaffold, of which
the thermostability of the immunogenic protein scaffold is not
compromised by foreign loop insertion.
[0009] Conception of a monospecific B-cell epitope and its
conjugation as synthetic peptide unto an immunogenic protein was
pioneered by Atassi, Lerner and Brown in the late 80's (Rowlands et
al., 1983, Nature, 306: 694; Atassi, 1978, Immunochem., 15: 909).
Most antigenic structure are presented as a loop constrained by the
secondary alpha helix and beta sheet structure, and properly folded
in the three dimensional array determined by favorable
energetics.
[0010] Through extensive studies of numerous potential B-cell
candidate epitopes, a B-cell loop antigenic epitope, taken out from
the native constrained secondary and tertiary protein folding, is
distorted in conformation. Such synthetic or recombinant peptides
randomly conjugated to or integrated to a protein carrier backbone
exhibited thermodynamically unpredictable, multiple distorted,
random conformations (Rowlands, et al., 1983, Nature, 306:694).
Synthetic or recombinant linear IgE B-cell epitopes without proper
constraint remain in a state of complex random array without
definable structural integrity. Constraining scaffold in supporting
the antigenic loop is required for enabling functional native
conformation with structural integrity.
[0011] In contrast, conception of constrained IgE B-cell epitopes
prompts the step to constrain the IgE B-cell epitope directly in
the thermostable protein scaffold, whereby functional native
conformation of the constrained neutralizing IgE B-cell epitopes
can be enabled by the constraint. The embodiment of this invention
further improves the constraining platform in placing B-cell
epitopes into the super constraining beta (b)-strands, and further
strengthened by the thermostable cystine knots, and finally
integrated onto another thermostable protein scaffold, engineered
in an optimal oxidative folding chemical milieu. Hence the
embodiment of the three improvements in this invention enables the
native expression and structural integrity of the B-cell
epitopes.
[0012] The embodiment of this invention with active,
conformationally constrained IgE B-cell epitope vaccine improves
over the passive neutralizing anti-IgE monoclonal antibody (Chang,
1995, U.S. Pat. No. 5,428,133): (i) Sustained circulating
IgE-Xolair complexes in treated patients cause long-term IgE
suppression. The regimen requires 36 to 54 week-long treatment in
order to neutralize 95% circulating IgE. However, due to the small
size of immune complexes, circulating IgE/IgG1 Xolair complexes
assume a half-life of 21 days of IgG1 (IgE lasts only one day);
consequently, total circulating IgE in the complexes are
persistently elevated .about.100 fold as a result of treatment
(Chang, 2010, Nat. Biotech., 18:157).
[0013] Active IgE B-cell vaccines embodied by this invention
improve the safety margin by producing active polyclonal anti-IgE
in the vaccinated recipients with appropriate length of protection
based on the vaccination/booster regimen. The duration is
controlled by reactivation of memory CD4 helper T-cells to the
protein scaffold. Due to the polyclonal antibodies, the clearance
of circulating and mucosal IgE and IgG complexes will be efficient
via the liver sinusoids and Kupffer cells. Furthermore, as a murine
human chimera antibodies, Xolair causes anaphylaxis in individuals
(3.14/1000 patients vs 5.4 events/million shots), the constrained
IgE B-cell active vaccine induces endogenous autologous anti-IgE
indigenous to the host.
[0014] (ii) Xolair is inefficient in targeting the mission-critical
pathogenic IgE in the lung. The passively delivered Xolair via the
subcut route, sieved through afferent lymphatics into thoracic duct
lymph into the general blood circulation, without permeating into
the critical sites of the lung, central for allergic asthma.
Allergen-specific IgE, secreted by IgE plasma cells present in
induced peribronchus-associated lymphoid tissues (iBALT), into the
surrounding lamina propria under the bronchial epithelial and
endothelial cells, remains inaccessible to circulating anti-IgE
monoclonal antibodies, whose delivery depends solely on
inflammation-mediated changes in vascular permeability (Lambrecht,
2009, Nat. Med., 31: 412).
[0015] A further embodiment of this invention is that IgE B-cell
vaccines delivered via mucosa route of immunization elicit anti-IgE
in iBALT that neutralizes pathogenic IgE in situ in the iBALT. The
FG super b-strands constrained IgE B-cell epitopes with or without
Min-23 cystine knot constraint, integrated onto the immunogenic
protein scaffold can be employed as IgE B-cell vaccines. B-cells
recognizing native, constrained FceRIa receptor-binding IgE-B-cell
epitopes are activated by CD4 helper T-cells reactive with CD4
helper epitopes on the immunogenic protein scaffold. Anti-IgE
antibodies of the IgA and IgG classes can be released directly in
mucosal secretion in addition to circulation via a preferred
mucosal route of immunization with FDA-approved adjuvants, Toll
like receptor (TLR)-7 agonist imiquimod, alum, lipid A-based
adjuvant or TLR-9 adjuvant presently being evaluated.
[0016] The designed constrained IgE B-cell vaccines elicit
polyclonal neutralizing anti-IgE of the IgG and IgA classes that
inhibits IgE-mediated mast cell degranulation, and prevents airway
inflammation and airway hyper-reactivity (Ahr) (Zuberi et al, 2000,
J. I., 164: 2667). Thus engineering constrained IgE B-cell epitopes
in the FG super b-strands and cystine knots, integrated onto the
immunogenic protein scaffold yields can lead to new anti-IgE
pan-allergy vaccines that can benefit asthmatics of different
disease spectra through mucosal IgE targeting and
neutralization.
SUMMARY OF THIS INVENTION
[0017] The embodiment of this invention is to identify super
constraining b-stranded secondary structure, and employing the
identified super b-strands to accommodate and support the newly
inserted a monospecific B-cell epitope of the loop structure, which
constitutes the active site for receptor recognition or
protein-protein interactions in the inflammatory pathways or
bacterial or viral infections. The robust or super energy-favorable
b-stranded secondary structure can next be integrated into a
thermostable cystine knotted structure onto a thermostable scaffold
protein. This presents a novel invented method of grafting and
preserving 3-dimensional B-cell epitopes for eliciting neutralizing
antibodies against microbial antigens and inflammatory
molecules.
BRIEF DESCRIPTION OF THE FIGURES
[0018] FIG. 1 depicts the primary sequence and secondary structures
of human IgE CHe2 to CHe4 domain. The secondary sequence was
assigned according to 1FP5 of the PDB, according to crystal
structure analysis of the human IgE-Fc epsilon3-Fc epsilon4
fragment by Wurzburg et al., 2000, Immunity, 13: 375.
[0019] FIG. 2 depicts expression of mini- and micro-IgE-GFPuv
fusion proteins. The mini-IgE domain (including C2-3 linker, BC, DE
and FG loops) and 4 micro-IgE domains (C2-3 linker, BCDEFG, DEFG
and FG) were inserted into the N-terminal of GFPuv to form
IgE-GFPuv fusion proteins. The fusion proteins were expressed and
analyzed by denaturing gels (A and B) and native gels (C and D)
against anti-GFP antibodies (A and C) and anti-human IgE antibodies
(B and D). Under denaturing conditions, all mini-IgE-GFPuv and
micro-IgE-GFPuv proteins had strong reaction with anti-IgE
antibodies (B). Two of the micro-IgE-GFPuv, DEFG-GFPuv and
FG-GFPuv, kept the strong reaction with anti-IgE antibodies under
native conditions (D). The sample loading orders were: 1. Human
IgE; 2. wtGFPuv; 3. pMini-IgE-GFPuv; 4. pC2-3-GFPuv; 5.
pBCDEFG-GFPuv; 6. pDEFG-GFPuv; and 7. pFG-GFPuv.
[0020] FIG. 3 depicts mapping super b-strands of FG microdomain
onto GFP. Synthetic oligonucleotides corresponding to various
truncated N- or C-termini of the human FG loop amino acid sequences
were ligated to the N-terminus of GFP.sub.UV (with c-His
constructed in the lab) by assembly PCR reactions. Suitable lengths
(-5AAN, and -10NAAN) were determined as minimal sequences for
expressing indigenous native human FG loop sequence, HLPR. The
diagram showed that the full length FG microdomain (amino acids,
413-439), including the FG core loop (HLPR) flanked by the b-strand
sequences (YQCRVT) and (LMPST), was prepared by PCR using the human
IgE heavy chain gene as a template, ligated by assembly PCR at the
N-terminus of GFP. Various 5' (N-) and 3' (C-) truncations were
performed with the arrow depiction, and the orientation of FG super
b-stands was depicted on both the N- and C-terminal of GFP.
[0021] FIG. 4 depicts mapping flanking b-strands of truncated C2-3
microdomain. The diagram showed that the full length C2-3
microdomain (amino acids, 308-345), including the C2-3 core loop
(NPRGVS) flanked by the b-strand sequences (TFEDST) and (AYLS), was
prepared by PCR using the human IgE heavy chain gene as a template,
ligated by assembly PCR at the N-terminus of GFP. Various 5' (N-)
and 3' (C-) truncations were performed with the arrow depiction,
and the orientation of truncated C2-3 are depicted on both the N-
and C-terminal of GFP as prepared in the single, bidentate and
bifunctional orientations.
[0022] FIG. 5 depicts reactivities with native FG loop B-cell
epitopes, flanked by FG super b-strands on the truncated FG
microdomain. Full length of FG and C2-3 were prepared with
oligonucleotides with HindIII cloning sites and recombined with
GFP.sub.UV. Site-directed mutagenesis (SDM) with different
truncations from N- and C-termini was performed with primers devoid
of different length of sequences. Recombinant clones were obtained
by high efficiency bacterial transformation, and recombinant
protein prepared by batch purification on the IMAC bead. To detect
the native conformation, the affinity pure FG-GFP and C2-3-GFP,
truncated as design were not boiled and the running sample buffer
did not contain 2-ME reducing agent, and 1% SDS substituted with 1%
CHAPS; the running buffer contains 0.1% CHAPS. The gel was
transferred in 0.5% methanol in 0.5% CHAPS and probed by
neutralizing polyclonal goat anti-IgE antibodies (pAb, of several
sources from the NEN, Clontech, and the Bethyl lab).
[0023] FIG. 6 depicts detection of swapped native human C2-3, BC,
and DE core loop sequences in super b-strands of truncated FG
microdomain on GFP. FG-5 and FG-10 truncated FG-GFP were prepared
as above. SDM was conducted with primers with foreign IgE B-cell
epitopes: core loop sequences from C2-3, BC and DE, while devoid of
the native FG loop sequences were performed by primer extension.
The resulting mutated clones with the replaced loop sequences were
ascertained by DNA sequencing. Panel A: various loop-substituted
recombinant products with C-terminal His-tag were affinity purified
by IMAC column. Immune reactivity with 100 ng native inserted
sequences was detected under native condition as described in
legend of FIG. 5 by neutralizing polyclonal goat anti-IgE
antibodies. The neutralizing antibodies were tested blocking human
IgE binding to the recombinantly produced FceRI, D2/D1 subunit.
Panel B: conventional western blot was also performed with the
above products run under denaturing SDS-PAGE with heated sample
treatment buffer containing 2-ME and SDS.
[0024] FIG. 7 A/B/C depict comparison native expression of swapped
C2-3, BC, DE loop B-cell epitopes in super b-strands of FG-N5
versus FG-N10 FG microdomain onto GFP scaffold. A complete set of
three IgE B-cell loop epitopes, C2-3, BC, and DE core loop
sequences were cloned into the FG-5 FG core loop swapped GFP, and
FG-10 FG core loop swapped GFP. In the native detecting conditions,
FG native loop and loop swapped-GFP recombinant products exist as
tetramers around 120 KDa, characteristic of GFP in native
tetramers. The effect of deletion of core loop sequences on the
integrity of GFP protein scaffold is evaluated (lane 11 and 12 of
Panel C)
[0025] FIG. 8. depicts rodent sequences for C2-3, BC, DE loop
swapped surrogate vaccines. A corresponding set of surrogate rodent
vaccine were prepared by replacing the homologous rodent sequences
with the native human FG core loop sequence: C2-3L (EPRGVI), BC
(DLAE) DE (NNATL), and FG (DFPK) loops were swapped into the human
super b-strands that flank the native human FG loop with the native
human HLPR sequence deleted. The recombinant proteins are detected
by rabbit anti-26.82 rodent IgE prepared in the lab.
[0026] FIG. 9 depicts anti-IgE elicited by FG-N-10-GFP blocking
human IgE binding to recombinant human FceRIa. Panel A showed a
standard IgE binding curve to recombinant receptors with OD signal
.about.1.2 at 50 ng/ml (the red open bar). Panel B showed diluted
sera from the FG-N-10-GFP immunized mice attenuated the human IgE
binding to D2/D1 FceRIa.
[0027] FIG. 10 depicts design of super b-strands in truncated FG
microdomain in Min-23 series, M-19 onto GFP scaffold. The Diagram
depicts the sequence of Min-23, and Min-18 prepared from EETI-II
GFP template, and the design of the various truncated FG super
b-strands and bidentate, tridentate constructs with amino acid
residue designation of the truncated length.
[0028] FIG. 11 depicts mapping N-terminal truncated FG microdomain
and truncated FG-microdomain scaffolded in Min 18/19 on GFP.sub.UV.
N-terminal FG with different N-terminal truncations was prepared
from the full length FG microdomain-GFP by deletion primer
extension via SDM. FG-Min-23 series, i.e., Min-19 (onto GFP,
counting the first glycine from the (gly)3 linker as the nineteen
residue of the Min-23 series), was prepared by addition primer
extension via SDM with the aforementioned FG-5, FG-10, and FG-15
into the Min-19 construct. PCR fragment of Min-23 (and other Min-23
series) was prepared from the EETI-II-GFP. Forward and reverse
primers in were added in a PCR reaction with GFPuv-His EET1 wt as
the template, inserted onto the HindIII site of the GFP.sub.UV.
Min-19 of the Min-23 series is further prepared with truncation of
loop 5 into C-FC and ligated to the (gly)3 linker onto GFP. SDM was
employed with primer extension to introduce the full length of FG
microdomain into the Min-19. N-5, N-10 and N-15 primers were
introduced by SDM and primer extension for truncating the FG domain
mutants in Min-19. Bidentate and tridentate N-10 FG in Min-19
mutants were prepared by addition in excess of PCR fragment of
HindIII-digested FG-10 in Min-19 to HindIII digested
GFP.sub.UV.
[0029] FIG. 12 depicts determination of different N-terminal
truncation on native FG loop expression in Min-19. Selective
recombinant products of the Min-23 series: Min-19 with inserted
loop of various lengths with terminal His-6 tag were purified by
IMAC beads, and examined on the native gel, and blot detected under
non-denaturing conditions.
[0030] FIG. 13 depicts comparative native expression of homo- and
hetero-(bifunctional) truncated FG and C2-3 construct. Panel A.
Under native detecting conditions, affinity pure single FG with
N-5, N-10 and N-15, and bidentate FG N-5, N-10 and N-15, tridentate
FG N-15 versus truncated C2-3, and heterodentate, bifunctional
truncated FG and C2-3 on N- and C-terminal of GFP were compared for
native expression of IgE B-cell epitope by neutralizing anti-IgE.
Panel B. The intensity of expression was scored from below
detecting levels (0.0-0.2) up to a nominal assignment of four,
i.e., that equivalent to native human IgE standard).
[0031] FIG. 14 depicts expression of loop B-cell epitopes of BC and
DE swapped into loop 1, 2, 3, 5 of wild type EETI-II. Two
overlapping forward and reverse primers, each partially
encompassing wild type EETI-II were synthesized, and annealed, and
filled in. PCR reaction was next conducted with short forward and
reverse primer (with the HindIII site), and the amplified full
length of EETI-II was digested with HindIII and annealed with
HindIII digested GFP.sub.UV vector, and bacterial clone selected by
DNA sequencing. BC and DE loop B-cell epitope insertion mutants are
prepared by addition primer extension with forward and reverse
primers encompassing the overlapping sequence of EETI-II and the
added BC, DE loop sequences via SDM.
[0032] FIG. 15 depicts native expression of BC and DE loop sequence
in loop 1, 2, 3, 5 of EETI-II wild type. To facilitate oxidative
folding for the formation of triple cystine bridge, EETI-II with
loop substitution was amplified by PCR with forward and reverse
primers with GFPuv-His EET1 wt as the template. Forward primer
started with gcggccgc of the Not 1 site and reverse primer with
gaattc of the EcoRI site. PCR product was digested with Not1/EcoR1,
and ligated to pMal5pE that was digested with Not1 and EcoR1 on the
C terminus of the maltose gene with the removal of the
Gly-Gly-linker from pMal, and the fused PCR fragment was then
cloned into Not1/EcoR1 pMal5pE. The series of recombinant EETI-II
wild type loop substitute-pMal products were expressed in
periplasmic space and purified by maltose column, and immune
reactivities evaluated with neutralizing anti-IgE under native,
non-denaturing conditions.
[0033] FIG. 16 depicts native expression of FG core loop in
truncated FG microdomain scaffolded in Min-18 subject to oxidative
folding. Min-18 of the Min-23 series was prepared as follows:
Forward and reverse primers were added in a PCR reaction with
GFPuv-His EET1 wt as the template with the synthesized product as:
Gcggccgc (Not 1)
CTAATGCGTTGCAAACAGGACTCCGACTGCCTGGCTGGCTGCGTTTGCGGGCCCA
ACGGTTTCTGCGGA (Min-23) gaattc (EcoRI). PCR product was digested
with Not1/EcoR1, and ligated to pMAL 5pE (NEN), that was digested
with Not1 and EcoR1 on the C terminus of the maltose gene with the
removal of the Gly-Gly-linker from pMal, and the fused PCR fragment
was then cloned into Not1/EcoR1 pMal5pE. Min-18 of the Min-23
series was then prepared by SDM with deletion primer of loop five
of Min-23, retaining only the phenylalanine (F). Next to obtain the
differentially truncated N- or C-mutants, overlapping primers with
omission of different N-terminal sequence of FG microdomain were in
SDM reaction Min-18-pMal, and the clone with the correct constructs
were ascertained by DNA sequencing.
[0034] FIG. 17 depicts native expression of C2-3 core loop in
truncated 2-3 microdomain in Min-18-pMal subject to oxidative
folding. The procedure is similar to that described in legend of
FIG. 16 with the SDM conducted with primer that prime that
initialize with Min-18 sequence with omission of 5' sequence of
C2-3 microdomain.
[0035] FIG. 18 depicts enhanced reactivities of native truncated
N-5 FG-Min-18-pMal (Min-23 series) with neutralizing anti-human
IgE. Truncated FG-N-5-Min-18-pMal was purified form periplasmic
space via maltose column. 150 ng of the recombinant constructs of
various deletions from the N- or C-terminus, including HLPR deleted
constructed were detected under native conditions with neutralizing
anti-IgE. The intensity of the expression was compared with an
equivalent dose of human myeloma IgE (BED). Notably, the full
length FG microdomain failed to express native FG loop, while the
intensity of native loop HLPR of one single B-cell epitope
expressed in FG N-5 construct in the oxidative periplasmic space,
exceeded that of myeloma human IgE containing all four IgE
neutralizing B-cell epitopes.
[0036] FIG. 19 depicts enhanced reactivities of native truncated
N-10 FG-Min-18-pMal (Min-23 series) with neutralizing anti-human
IgE. Truncated FG-N-10-Min-18-pMal was purified form periplasmic
space via maltose column. 200 ng of the recombinant constructs of
various deletions from the N- or C-terminus, including HLPR deleted
constructed were detected under the native non-denaturing
conditions with neutralizing anti-IgE. The intensity of the
expression unpurified, in the bacterial extract, was compared to
high dose of human myeloma IgE (BED). Notably, full length FG
microdomain exhibited weak expression of native FG loop in the
bacterial extract, while the intensity of native loop HLPR of one
single B-cell epitope expressed in FG N-10 construct in bacterial
extract in the oxidative periplasmic space, exceeded that of
myeloma human IgE with all four neutralizing B-cell epitopes.
[0037] FIG. 20 depicts reactivity of native versus denatured
truncated C2-3 of 22 amino acids in Min 19-pMBP. Different lengths
of truncated C2-3 in Min-18-MBP constructs were prepared by SDM by
primer extension. Recombinant products were affinity pure on
maltose column, and 100 ng purified products were evaluated under
both native and denaturing conditions. The construct of 22 amino
acids from the C-terminus of the C2-3 microdomain was detectable in
both native and denatured conditions. Moreover, C-terminal amino
acids were critical for the native expression of FG loop B-cell
epitopes, and C-7 constructs (both C-7 and C-7/N-15 constructs)
failed to exhibit the indigenous FG loop B-cell epitopes.
DETAILED DESCRIPTION OF THE INVENTION
[0038] IgE-mediated allergic asthma and allergic inflammation
affects 46 million US population and 300 million worldwide.
Research on IgE molecules for a cure for IgE-mediated allergic
diseases has been intense for decades. Spatial IgE receptor-binding
B-cell epitopes defined according to X-ray IgE receptor-cocrystal
(Garman et al., 2000, Nature, 406: 259), were engineered via the
constraining platform designed by the lab. There is yet no active
vaccine prepared based on preserving neutralizing IgE B-cell
epitopes based on constraining secondary structure on thermostable
protein scaffold. Vaccine-elicited antibodies to well-defined
neutralizing IgE B-cell epitopes can prevent or neutralize IgE
binding or sensitization to type I IgE Fc receptors (FceRIa).
[0039] Because IgE, complexed with vaccine-elicited anti-IgE is
sequestered from binding to FceRIa, the immune complexes can be
cleared from circulation or IgE-infested mucosal sites of the lung
and the GI tract. The embodiment of this invention will also not
cause cross-linking receptor bound IgE on sensitized mast
cells/basophils since the IgE B-cell epitopes, which the
neutralizing antibodies recognize, are blocked from interacting
with the FceRIa receptor binding sites. Thus IgE sensitized mast
cells/basophils can not be activated by neutralizing antibodies and
undergo mast cells/basophil degranulation.
[0040] The feasibility of retaining the reconstructed, constrained
three-dimensional B-cell epitopes was demonstrated by its capacity
of being recognized by neutralizing antibodies under the native
non-denaturing conditions. The embodiment of scaffolding with two
to three successive, categorically different constraining molecular
devices has not been undertaken. Straight forward synthetic peptide
coupling to protein carrier led to random and/or linear peptide
B-cell epitopes, and antibodies raised to the linear peptide
epitopes do not cross-react or cross-neutralize native IgE
proteins, nor host inflammatory cytokines, nor cross-react and
neutralize pathogenic bacteria or viruses.
[0041] Although the knowledge exists concerning IgE loop sequences
that directly bind to FceRIa by X-ray cocrystal, or secondary
structures adjacent to the receptor-binding loop of IgE (Garman,
2000, Nature, 406: 259), there exists no enablement of translating
the receptor-binding, loop antigenic sequences into B-cell vaccines
that elicit neutralizing antibodies to the receptor-binding sites
of IgE. The embodiment of the invention enables the native IgE loop
sequences immunogenic for eliciting IgE neutralizing antibodies as
protective allergy vaccine.
[0042] IgE in solutions maintains a closed dyad symmetry, slightly
twisted for 3.degree. at the C2-3 junction. This twisted angle
ensures an asymmetric docking of IgE dimer on receptor molecules.
Herein, we exemplify the two receptor-binding sites of IgE. The
exemplification serves a heuristic attempt to describe the need for
a constraining strategy for accommodating the receptor-contacting
loop structure onto appropriate constraining scaffolds: b-strands
or super b-strands, cysteine knots, and thermostable protein
scaffold in toto serving as scaffolding for the IgE
receptor-binding sites. Not only the receptor-binding sites but
also the adjacent sites to the IgE receptor-binding sequences, to
which interfering antibodies can be raised, serve as the druggable
sequences for eliciting IgE neutralizing and/or receptor
interfering antibodies. The embodiment includes the constraining
both FceRI-receptor binding and receptor-interfering IgE sequences
in super b-strands and/or cysteine knots as first and second-tiered
constraining scaffold onto further a third-tiered thermostable
protein scaffold.
[0043] X-ray structure of IgE/receptor complexes (Garman et al.,
2000, Nature, 406: 259) showed that receptor D2 and D2-D1 linker
exhibit asymmetric contact to different amino acid residues on both
half molecules of IgE. There are two major recesses in the IgE
receptor: the P426 and Y131 pockets. The BC, DE, C2-3L loops of one
half IgE molecule, bind predominantly to the Y131 pocket over an
830 angstrom.sup.2 surface, whereas four main amino acids of the FG
loop (HLPR) on the other IgE half molecule, bind the P426 pocket
(named after the HLP (426) R), spanning the D2 and D2-D1 linker of
FceRIa. The P426 receptor pocket buries a large surface area of IgE
predominantly the FG loop about 970 angstrom.sup.2.
[0044] Surface areas of FG and antigenicity: The contact area of
crucial FG loop of IgE with receptor's BC loop and D2-D1 linker
covers a major stem area .about.670 angstrom.sup.2 of P426, and the
C2-3 linker contributes at the receptor's FG loop tip area to
.about.300 angstrom.sup.2 of P426. The four amino acids, HLPR form
intimate contact with about 450 angstrom.sup.2 out of a total of
930 agstrom.sup.2 space (the rest occupied by C2-3L), and the R
residue of FG loop also buried deep in the Y131 pocket.
[0045] Overall, two asymmetric amino acid sequences of the dimeric
CHe2-CHe3 of IgE heavy chain work in synergy to bind to the
receptor D2 and D2-D1 linker to confer high affinity IgE binding to
receptor in a one to one stoichiometry. The contact IgE residues to
receptors are respectively C2-3L, BC loop, DE loop, and FG loop.
The surface IgE receptor-binding loops are antigenic and solvent
accessible according to B-cell epitope predictive algorithms,
suitable for neutralizing antibody targeting. While all four FG
loop core residues of one IgE heavy chain, i.e., HLPR, bind to the
P426 pocket, the H residue of the FG loop and C2-3, BC and DE of
the other IgE chain bind to the Y131 receptor pocket. Initially,
the FG loop of IgE and predominantly the C2-3, BC and DE loops bind
to a single receptor domain, the Y131 pocket, resulting in a low
affinity interaction (10.sup.5 M.sup.-1) (Robertson, 1993, J. B.
C., 268: 12736). Subsequently, engaging the entire FG loop (HLPR)
to D2-D1 linker of the P426 pocket along also with the C2-3 loop of
IgE, and FG and C2-3 loops in concert, renders a high affinity
binding to IgE (10.sup.9-10 M.sup.-1).
[0046] The binding energetics is the depiction of the energy
landscape of the IgE ligand binding to the receptor, an integrated
expression of enthalpy and entropy of the binding, which controls
the folding of the respective contact of the loops between the
D2-D1 domains of the FceRI receptors and IgE ligand. Thus to take
the amino acids out of the context of the native constraining
milieu without imposing a substitute constraining device or
devices, i.e., preparing synthetic peptides onto KLH, BSA or viral
like particle conjugates via direct chemical conjugation at the
random locations of carrier proteins, or the straightforward use of
two flanking cysteine linearly, leads to the loss of the native
conformation.
[0047] One-step or single constraint for retaining native B-cell
epitopes: Previously, the construction of monospecific neutralizing
IgE B-cell epitopes in a special constrained format in the internal
sequences of the GFP protein, which simultaneously also serves as
the protein scaffold has been advocated (Chen, 2008, J. Immunol.
Meth., 333:10). In this single-step constraint, the foreign loop
epitopes are inserted or replacing the endogenous loop of the
protein, which also serves as the scaffold for exhibiting the
inserted sequences. The demand of the scaffold protein to perform
is two-fold in accommodating the inserted determinants as well as
in serving as an overall protein scaffold.
[0048] Two- to three-step, or multiple constraints for retaining
B-cell epitopes: the embodiment of the invention. To constrain the
above epitopes, a constraining platform is required. This prompts
the invention of the first tier in seeking a stable pair of
b-strands within the four receptor-binding loops of IgE. A series
of truncation mutants of the four receptor-binding IgE segments
(C2-3, BC, DE, and FG) were prepared unto GFP, and the native
epitopes were expressed and test with neutralizing anti-IgE under
native conditions, devoid of reducing agents, heating, and
denaturing SDS by native western blots. The core embodiment of this
invention is to determine the existence of such a super b-strands
which can serve as a universal clamps for accommodating as many
pertinent loops to the native state critical for invoking a
pharmacological response, or serving as the antigen or vaccine for
eliciting neutralizing antibodies against IgE molecules or
proinflammatory cytokines, or blocking bacterial or viral
infectivity.
[0049] The embodiment of this invention resides in enabling a
super-stable loop epitope (preferably with the proline as a kink)
with a flanking super b-stranded scaffold. The embodiment of the
invention is to invoke the IgE B-cell vaccines with one of the four
FceRIa contacting IgE B-cell epitopes as monospecific vaccine
alone, and in combination as a combinatory polyvalent vaccine.
Taken together, we therefore reason that antibodies to a properly
designed FG loop and other receptor-binding or receptor-interfering
sequences can similarly efficiently prevent IgE binding to receptor
via direct blocking or steric hindrance, due to blocking on one
half molecules at the P426 pocket, and in synergy with blocking
Arg.sup.427 of the other half molecule at the Y131 pocket.
Similarly, constrained C2-3 loops in super b-strands and/or
cystine-knotted on a thermostable, immunogenic protein scaffold,
elicit neutralizing anti-IgE that block IgE binding to receptor at
the Y131 and P426 sites. DE and BC core loop sequences constrained
in super b-strands and/or cystine-knotted onto protein scaffold,
elicit neutralizing antibodies that block at the Y131 sites. Direct
blocking at one IgE ligand binding site causes also steric
hindrance at the P426 and vice versa.
[0050] Receptor-blocking neutralizing antibodies elicited from
properly scaffolded receptor-binding or receptor interfering IgE
sequences protect human IgE from binding to human high affinity IgE
receptors on mast cells, basophils, dendritic cells, neutrophils
and eosinophils.
[0051] Thus, the descriptive sequence knowledge based on X-ray
cocrystal is directly enabled into a three dimension preserved
conformation by the embodiment of this invention, and is translated
into a pharmaceutical product for treating IgE-mediated allergic
diseases. Specifically shown in Examples: (i) With regard to
specific FG loop, we showed that the FG loop is naturally, most
robustly scaffolded within its flanking b-strands, which is also
capable of super performance in that the b-strands of the FG loop
also constrains core loop sequences of BC, DE and C2-3L loops.
[0052] (ii) It is well known that the cystine stabilized b-sheets
(CSB) containing miniprotein, cystine knots (CK), exhibits a broad
range of bioactivities and are exceptionally stable
(melting/denaturing temperature (Tm>100.degree. C.) being
resistant to chemical, thermal and enzymatic degradation.
Therefore, the FG loop with the native b-stranded scaffold can be
further scaffolded in the cystine knot stabilized b-sheets. (iii)
The doubly constrained FG loop can co-fold with GFP protein
(Tm.about.100.degree. C.) that supports the overall cystine
knots-constrained FG loop as well as activating helper T-cells
required for antibody production as shown in our lab and affinity
maturation. The fluorescence of GFP also serves as a first
indication for the integrity of the fusion protein.
Candidacy of Linear B-Cell Epitopes as Neutralizing B-Cell
Vaccines
[0053] Conception of monospecific B-cell epitope and its
conjugation as synthetic peptide unto an immunogenic protein was
pioneered by M. Atassi, Richard Lerner and Fred Brown in the 80's
(Atassi, 1978, Immunochem., 15: 909; Rowlands, 1983, Nature, 306:
694). However through extensive studies of numerous potential
B-cell candidate epitopes, B-cell epitopes randomly conjugated to
protein carriers are thermodynamically unpredictable, and
exhibiting in a random, distorted conformation unlikely to present
the native antigenic epitopes, resulting in frequently linear
epitopes recognized by the denatured PAGE/western blot condition by
anti-peptide antibodies (Maloy, 2012, Curr. Prot. Immunol. Unit
9.4).
[0054] The linear epitope is likely to represent a minor folding
pathway of B-cell epitope presentation similar to the presentation
of the fraction of denatured protein co-existing with the native
protein. The other source of anti-peptide antibodies is derived
from the degraded products of the vaccine, which assume linear or
amino acid sequence dependent B-cell epitopes recognized by the
host antibody repertoire. This embodiment of the invention disables
generating antibodies against the pool of spurious
sequence-dependent linear epitopes, which are of insignificant
import in serving as prophylactic or anti-inflammatory antibodies.
The anti-linear B-cell epitopes antibodies can serve a minor role
in clearing the effete life cycle products of degraded IgE,
inflammatory proteins, cytokines, protein kinases, transcription
factors. While denatured B-cell epitopes of the viral and
inflammatory proteins can be cleared by the anti-peptide
antibodies, these antibodies do not neutralize active ongoing
bacterial and viral infectious agents, nor neutralize inflammatory
protein, IgE and cytokines to achieve therapeutic effect.
[0055] Although anti-linear peptide antibodies are dominant in the
reagent markets, this approach of random conjugation of B-cell
epitopes to proteins is rarely relevant for the purpose of
preparing protective vaccines. For decades randomly coupled
synthetic peptides has not led to anticipated B-cell vaccine
candidates in order to elicit protective neutralizing antibodies,
although linear peptides are considered CTL vaccine candidate since
class I MHC typically accommodates linear peptide sequences, and
not native peptides (Fridman et al. 2012, Oncolmmunol, 1:
1258).
[0056] Chemical conjugation of IgE B-cell epitopes to protein
carriers without constraint, invariably leads to distorted
conformation of the epitope, or in an extended linear display,
which was detected in SDS extended linearized form detected by
antibodies to IgE synthetic peptides randomly coupled to the
protein carrier. Chemical coupling reagents including homo- and
hetero-bifunctional reagents such as MDS and SPDP, and random
chemical coupling reagents such as carbodiimide and glutaraldehyde
have been deployed for raising only anti-linear peptide antibodies.
And there are numerous conjugating compounds developed by the
reagent companies, Sigma, and Pierce. Random chemically conjugated,
unconstrained B-cell epitopes onto the Qbeta structural protein in
a viral like particle (VLP), an platform initially developed for
eliciting linear CTL epitopes are subject to distortion of the
native conformation (Bachmann et al, 2002, U.S. Pat. No.
7,128,911).
[0057] Embodiment of this invention enables active site specific
viral B-cell vaccines: Structural vaccine design pertaining to
B-cell vaccine epitope is gaining increasing importance in major
viral infectious diseases, i.e., HIV (Johnston and Fauci, 2007, N.
Eng. J. Med., 356: 207). The visualization of the protective
surface by b12 and ARC01 elucidates both the CD4 binding site
(CD4bs) of gp120 (interacting with host CD4 molecule) and a
mannose-binding site surface. To form a mimetics for a large
complex surface or carbohydrate-binding site via protein fragments
or genetically modifying the whole antigenic surface via global
antigen resurfacing has met with major computation challenges. The
embodiment of this invention indicates that critical fragments of
contact residues of CD4bs may be inserted into the super b-strands
and the cystine knots on the support of a protein scaffold.
[0058] Gp41 is a subunit of the envelope protein complex,
non-covalently bound to gp120 and provides a second step for HIV's
entry to the cells via contacting host cell CD74 (Zwick at al.,
2001, 75: 10892). Thus blocking gp41 with neutralizing antibodies
can attenuate viral infections. The invariant 30 amino acids of
gp41 of HIV in the membrane proximal region can interact with CD74
and cause enhanced infectivity. In the embodiment of the invention,
CD74 contact loop epitopes can be inserted into the super
b-strands, in the cystine knots on the thermostable protein
scaffold.
[0059] In the influenza virus, the hemagglutinin binding the sialic
acid accounts for infectivity. The two glycoproteins of the
influenza virus membrane, hemagglutinin (HA) and neuraminidase
(NA), both recognize sialic acid (Gamblin and Skehl, 2010, J. B.
C., 285: 28403). Initiation of virus infection involves multiple
HAs binding to sialic acids on carbohydrate side chains of
cell-surface glycoproteins and glycolipids. Following virus
replication, the receptor-destroying enzyme, NA, removes its
substrate, sialic acid, from infected cell surfaces so that newly
made viruses are released to infect more cells. Both activities are
the targets of antibodies that block infection. The embodiment of
this invention is to place the core loop region of neuraminidase in
the super b-strands (FG), in the cystine knots on a scaffold
protein.
[0060] The binding depression surrounds the sialic acid domain with
three primary regions of the hemagglutinin structure. This region
consists of a loop-helix-loop (130 loop-190 helix-220 loop)
structure forming the triangular opening into the beta-sheet
depression. The core loop regions, i.e., the 130 loop and 220 loop
are accessible to the aqueous phase in triplicates and are target
for loop-specific mono-specific neutralizing antibodies. In the
embodiment of this invention core loop sequence of 130 and 220
loops can be inserted in the super-b-strands (FG) in the cystine
knots on a protein scaffold as HA-specific B-cell vaccines.
[0061] Despite the knowledge of three dimensional structures of
Flu, RSV and HIV protective proteins, active vaccines with
monospecific neutralizing B-cell epitopes to protect against viral
infections are not forthcoming with synthetic peptides conjugated
to protein carriers. The embodiment of FG loop, and BC, DC, C2-3
linker loop-specific vaccine in a highly constrained super
b-strands on a stable protein scaffold, can be extended to
similarly molecular engineered active site (such as FG
loop)-specific active microbial vaccines such as GP120, and
GP41-specific active HIV vaccine, human HA-specific flu vaccine,
and human RSV vaccines (Dudas and Karron, 1998, Clin. Microbiol.
Rev., 11: 430).
[0062] Embodiment of this invention as a remedy for linear B-cell
epitopes: The effort herein is to invent a general antigen display
via a combined effort to include scaffolding the B-cell vaccine
candidate loops into the indigenous super b-strands of truncated FG
microdomain, with or without further constraint into the
thermostable cystine knots, integrated onto the thermostable
protein scaffold.
[0063] This approach enables a new platform for discovery of the
critical monospecific B-cell vaccines for major IgE-mediated,
cytokine-mediated inflammatory diseases, and major viral infectious
diseases. Approach of random synthetic peptide conjugation to
protein carrier leads to linear peptide epitope presentation. The
embodiment of this invention enables the grafted B-cell epitopes to
assume the native, three dimensional antigenic B-cell neutralizing
epitopes
[0064] The embodiment of the invention enables treatment of human
IgE-mediated allergy therapy: Specific immunotherapy (SIT). SIT is
an FDA-approved prevalent therapy, based on induction of specific
anergy by regulatory CD4 T-cells and immune deviation of CD4
T-cells. Extracts of allergenic source materials have been
employed, which require safety supervision.
[0065] The embodiment of this invention for preparing FG, C2-3, BC
and DE loop monospecific and polyvalent IgE B-cell vaccines in FG
super b-strands offers expediencies over productions and clinical
testing of a host of recombinant allergens. The IgE loop-specific
B-cell vaccine ensures the safety in contrast to crude allergen
extracts. As a pan-IgE neutralizing vaccine, it covers diverse
allergen specificities, and its efficacies and safety can be
evaluated by measuring the protective anti-IgE loop in contrast to
immune deviation and induction of allergen-specific regulatory T
cells (Treg).
[0066] By targeting the receptor-binding and receptor interfering
sequences of human IgE, a commonly shared antigenic epitope, this
active vaccination with conformational constrained IgE B-cell
active vaccine can alleviate a wide spectra of IgE-mediated
diseases caused by a myriad of allergens. The cost/benefit ratios
of the FG loop vaccine over passive monoclonal antibodies are
favorable in extending the patient base. Vaccination via mucosal
immunization can achieve protection at the mucosal organs, lungs
and the GI tracts. The safety of the active vaccine will be ensured
by a regimen for booster-required six month-treatment duration
windows similar to that of the passive anti-IgE antibodies.
[0067] One aspect of embodiment of the invention resides in
immunogenicity of the protein scaffold in controlling the duration
of anti-B-cell epitope response. The protein scaffold that supports
monospecific B-cell epitope constrained by the super b-strands with
or without further constraint of the cystine knots, can recruit CD4
helper T-cells that activate B-cells specific for native IgE B-cell
epitopes. The bifunctional protein scaffolds include but are not
limited to green fluorescent protein and maltose-binding protein.
The longevity of anti-IgE responses can be moderated by a vaccine
booster regimen. Without a booster dose, CD4 helper T-cells become
quiescent and neutralizing anti-IgE responses decline and basal
levels of IgE resume. The recovered levels of autologous IgE also
cause tolerance of IgE-specific B-cells. These processes ensure
that no persistent anti-IgE responses cause long-term suppression
of circulating IgE or mucosal IgE. The embodiment of this invention
also can sustain high titers of neutralizing anti-bacterial and
viral antibodies with vaccine boosting for activating memory CD4
helper T-cells specific for protein scaffold.
[0068] Another embodiment of the invention enables treatment of
IgE-mediated allergy in pet animals and economically useful large
animals
[0069] Because of the identity of the FG loop core sequence among
humans and non-human primates, it efficacies and safety can be
further tested in these species. Veterinary IgE B-cell vaccines can
be prepared by replacing the native FG loop sequences of human FG
super b-strands on a protein scaffold with FceRI-binding IgE
sequences of feline, canine, equine, and bovine species.
EXAMPLES
Example 1
Antigenicity
Preservation of Constrained Human IgE Mono-Specific Subunit Vaccine
Epitopes
[0070] The super b-strand flanking sequences and the replaced
loops: The major embodiment of the invention is to enable the most
rigid b-strands energetically favorable for accommodating the
foreign insertion loop epitope. The discovery step consists of
determining a robust IgE FceRIa binding B-cell epitope region or
microdomain that can be molecularly presented by a thermostable
protein scaffold. Following this identification, the region or
microdomain can be further trimmed to identify the critical rigid
secondary structure, flanking b-strand that present the endogenous
and swaps with foreign loop epitopes.
[0071] FIG. 1 describes sequences from the primary amino acid
sequences derived from human IgE constant regions sequences 1FP5 of
the PDB database. The sequences of interest are from the four high
affinity IgE receptor-binding regions and the core loop sequences:
C2-3 region is defined as RTYTCQVTYQGHTFEDSTKKCADS (NPRGVS,
332-327) AYLSRPSP (308-334) cloned into the GFP for truncation for
the minimal sequences required for presenting native IgE B-cell
epitopes. NPRGVS (332-337) is the core loop sequences that contact
the IgE FceRIa. DLAPS (362-366) is the receptor-contacting core
loop sequence of the BC region. RNGT (393-396) is the
receptor-contacting core loop sequence of the DE region.
[0072] TRDWIEGETHP(HLPR)TKTSGPR (408-440) is the FG region. The
core loop sequence (HLPR) is flanked by two b-strands as also
scaffold that can present the native FG core loop sequence HLPR,
and also can present core loop sequences of C2-3, BC or DE core
loop sequences for native B-cell epitope presentation.
[0073] Discovery of this embodiment consists of three enablement
steps: (i) selecting an intrinsically robust scaffold; (ii)
delineating the minimal length of the flanking amino acids for the
loop epitope; (iii) replacing the native loop epitope with foreign
epitope.
[0074] Enablement of super b-strands of the FG microdomain:
The selection of an intrinsically stable IgE epitope was first
dissected by expression the respective C2-3, BC, DE FG region (or
microdomain) and/or a contiguous C2-3/BC/DE/FG complete region
(minidomain) on the N-terminus of GFP, and the immune reactivities
to neutralizing anti-IgE under native conditions were
evaluated.
[0075] As shown in FIG. 2, the complete region of C2-3/BC/DE/FG
mini-IgE domain is under-expressed according to GFP under both
native and denaturing conditions (lane 3, Panel A and Panel C),
while IgE reactivities were noticed under denaturing conditions but
not under native conditions, indicating the folding of mini-IgE
domain negatively affects the conformation of GFP, and despite the
preservation of linear denatured B-cell epitopes, the critical
native IgE B-cell epitopes were however not expressed. In contrast,
C2-3 microdomain expressed on N-terminus of GFP caused strong
expression of GFP detected by both denaturing and native
conditions, indicating favorable GFP folding and expression (lane 4
of FIG. 2 Panel A and B).
[0076] Despite the favorable GFP folding in both native and
denaturing conditions (lane 4, Panel A and C) and detection of the
C2-3 B-cell epitope strongly under denatured conditions (lane 4,
Panel B), C2-3 epitope was non-detectable under native conditions
(lane 4, panel D). This indicates that C2-3 folding appears
restricted only to the native chemical milieu of IgE molecules, and
the native conformation is lost upon cloning onto GFP protein
scaffold. This further indicates that the b-strands flanking the
C2-3 are incapable of sustaining the presentation of the indigenous
C2-3, and suggests that this may not sustain the conformation of
the molecularly grafted foreign B-cell epitopes, i.e., a candidate
for the super-b-strands that can accommodate promiscuously a
diverse B-cell antigenic loop epitopes.
[0077] Next, C2-3 deleted from the mini-IgE domain also leads only
to expression of the linear epitope detected under denaturing
conditions (lane 5, Panel B). Importantly, further truncation of BC
in addition to C2-3 leads to strong augmented expression of GFP
under both denatured and native conditions, indicating the dual
microdomain construct does not affect the conformation of GFP (with
detected fluorescence), and importantly leads to the expression of
immune reactive DE and FG B-cell epitopes strongly under both
denatured and native conditions. Lastly, the single microdomain FG
construct caused also strong expression of native GFP conformation
(lane 7, Panel C) and the native expression of FG loop determinant
(lane 7, Panel D). Therefore the discovery of the robust chemical
structure, the super b-strands in FG microdomain, which constrains
the core FG loop, serves as the foundation for its use as a key
conformation constrainer for not only FG core loop but also core
loops of other IgE microdomain IgE, and extending to protective
B-cell epitopes in microbial infectious diseases.
Delineation of the Super-b-Strands in the Truncated, Minimal FG
Microdomain
[0078] 1. Strategy of Constraining FG Loop Antigen onto GFP
Scaffold
[0079] To determine the super-b-stands as scaffold, truncation was
made on the N-terminal ends of both C2-3 and FG segments. This
construct strategy consists of two molecular matrix layers of
super-beta strands, integrated with a thermostable protein
scaffold. FIG. 3 diagram showed that the design construct of FG
microdomain at the N-terminus of GFP scaffold, with deletion of the
microdomain from the N- and C-terminus. FIG. 4 showed molecular
integration of C2-3 microdomain into GFP scaffold, with truncations
from the N- or C-terminus.
[0080] We test whether the FG loop may be delineated and its
antigenicity studied by co-folding at the N-terminus of GFP. The
total FG loop (413-439), including the FG core loop (THPHLPR)
flanked by the b-strand sequences (YQCRVT) and (LMPST) was prepared
by PCR using the FG-GFP template or human IgE heavy chain cDNA as
templates, and then ligated at the N-terminus of GFP by assembly
PCR. In order to assess the native FG determinants, samples were
neither heated nor treated with 2ME, and separation and transfer
were conducted in native buffer, substituting SDS with CHAPS. FIG.
5 showed that N-terminal deletion of five, ten and fifteen amino
acids, did not affect the expression of the core FG loop epitope
(lane 7, 8, 9), and provided three potential enablement constructs
of inserting foreign B-cell epitopes to replace the native core
loop of the FG loop.
[0081] In contrast, as shown in FIG. 5, C2-3 is not capable for
providing the b-scaffolding device in that neither the full C2-3
microdomain nor its trimmed constructs: N minus ten, or fifteen or
twenty amino acid deletion eliminates the native immunoreactivity.
The full length DE microdomain augments reactivity with
neutralizing anti-IgE (compared to Lane 6, Panel D of the previous
FIG. 2). In one aspect of the embodiment of this invention, DE
microdomain integrated with GFP, can be similarly truncated for
determining presentation of the native DE loop sequences, and
extended to FceRI interfering sequences other than FceRIa receptor
binding sequences of IgE. The evidence indicates that the native
b-strands flanking the FG loop are the most robust b-strands
amongst four receptor-binding loops due to its reactivities under
native conditions to the polyclonal neutralizing anti-IgE. The
polyclonal goat anti-IgE neutralizing antibodies blocked IgE
binding to solid phase FceRIa receptors.
[0082] 2. Swapping the Foreign B-Cell Antigenic Epitopes Among the
Super-b-Strands onto GFP Protein Scaffold
[0083] The embodiment of this invention is therefore to utilize the
rigidity of the original native b-strands that flank the antigenic
loop in a b-hairpins or super b-strands may serve as a first order
constraining molecular clamps not just for the indigenous
sequences. And thus this leads to the inventive concept that
foreign loop sequences replacing the endogenous loop sequence,
i.e., the FG core loop sequences can also maintain the necessary
conformation of the super-b-strands, which in turn can constrain
the foreign replacement loops, and the overall productive folding
of the foreign B-cell loop in the b-strands can also help the
folding of the supporting protein scaffold.
[0084] In order to ascertain the specificity of FG loop sequence
detection by neutralizing anti-IgE, the native loop sequence is
deleted from the N-5 FG construct. As shown in FIG. 6, importantly
the elimination of the loop antigenic epitope removes its specific
reactivity to the neutralizing anti-IgE (lane 4, panel A). This
indicates that this precise location being the antigenic sites
being accommodated by the flanking b-strands is confirmed, and can
serve a site for exchanging or swapping with other foreign loop
sequences.
[0085] FIG. 6 also confirmed that with FG microdomain derivatives
with N-5 and N-10 deletion are strongly reactive to detected by
neutralizing anti-IgE under native conditions (lane 2 and 3, Panel
A, FIG. 6), while the FG loop with the core loop sequence (HLPR)
deleted was not detected (lane 4, panel A of FIG. 6).
[0086] The conformation of the FG loop is robust. It is possible
that both the flanking b-strands of the FG loop (YQCRVT; LMPST) and
the proline 422 as a kink of the THP (422)HTLP core loop sequence
work in synergy for forming this extra-stabilized hairpin FG loop.
The grafting of other loop sequences into the FG b-stand
scaffolding clamps renders it a robust central platform for
preparing future multivalent neutralizing IgE epitopes as the
vaccine. The conception of this invention is validated and
materialized by replacing the native FG core loop sequences with
three other IgE B-cell epitopes, C2-3, BC, and DE epitopes. As
shown in FIG. 6, it is of critical importance that this strategy of
accommodating the other there human neutralization sequences into
the deleted core residues that lead to restoration of the filled-in
swapped sequences: RNTL (the DE loop core sequence, lane 5), NPRGVS
(the C2-3L loop core sequence, lane 6) and DLAP (the BC loop core
sequence, lane 7) by native western to pAb under native conditions,
and BED IgE as positive control (Ln 8).
[0087] FIG. 6A showed that the FG-GFP with N-5, and N-10
truncations, or with various swapped loops was detected under
native conditions as a native tetramer of 120 KDa by neutralizing
pAb: polyclonal goat anti-IgE neutralizing antibodies. It should be
pointed out that since GFP, under native conditions, is present as
a dimer or tetramer by the X-ray data (1GFL, PDB bank), the
detectable FG loop swapped sequences in GFP protein scaffold
migrated at the 120 KDa. In contrast, these recombinant
IgE-epitopes GFP constructs also reacted with anti-GFP with the
corresponding 35 KDa band under denatured conditions, shown in
similar order in FIG. 6B. We have since then focused on the
C2-3-GFP and FG-GFP constructs due to their respective important
role in docking to high affinity IgE receptor; in particular the FG
loop appeared to bind to neutralizing antibody, Xolair according to
the computer fitting, epitope docking model (Zheng et al., 2008,
B.B.R.C., 375: 619).
[0088] The expression of GFP moiety in the above constructs is
compared. The same material, FG loop (-5N) and (-10N) was detected
as a 35 KDa band with anti-GFP under denatured conditions (lanes 2,
3 and 4, Panel B), while the empty FG loop was detected with less
intensity by anti-GFP, indicating that the presence of the native
endogenous sequence appears to stabilize the expression of GFP
scaffold, and/or the presence of the native loop sequence not only
prevents the distortion of the FG microdomain, but the integrity of
FG micro-domain in turn also supports the folding and integrity of
GFP. Thus protein folding properly consummated is dependent on
integrity of the secondary b-strand structure, which flanks the
endogenous loop sequences.
[0089] Although C2-3L can be constrained within the internal loop
of GFP (Chen, 2008, J. Immunol. Meth., 333: 10), it is not amenable
to other loop sequence insertions. Thus reproduction of all four
FceRI binding IgE B-cell epitopes by swapping and replacing the
native FG loop sequences with other IgE B-cell epitopes, indicates
strongly that the FG loop flanking b-strands constitutes the super
b-strands for scaffolding pharmaceutically important B-cell epitope
loop sequences, including receptor-binding IgE B-cell epitopes.
[0090] 3. Further Validation of the B-Cell Epitope Swapping in the
Super b-Strands of FG Domain
[0091] In yet another verification and extension of the embodiment
of the invention with loop sequences swapped in dual vectors, the
swapping of BC, DC and C2-3 core loop B-cell epitope is replaced in
both N-5 and N-10 super-b-strand scaffold of the FG microdomain.
The comparison of native gel reactivity with neutralization is
performed with loading with 200 ng of purified recombinants
products via the His-tag on the C-terminal of GFP scaffold.
[0092] As shown in FIG. 7A, the full length DE loop microdomain on
GFP protein scaffold maintains the native conformation reactive to
neutralizing anti-IgE (lane 3, FIG. 7A). This confirms the previous
observation that DE/FG-GFP construct maintains the stronger
reactivity than that of FG microdomain alone integrated in GFP
(lane 6 versus 7, Panel D of FIG. 2). This strongly suggests that
b-strands of DE microdomain may serve as another set of super
b-strands for accommodating other B-cell loop epitopes. A critical
important point is that N-5 and N-10 FG truncated constructs can
serve in concert to optimize accommodation of the three IgE core
loop B-cell epitopes selectively. Thus N-5 construct accommodates
optimally for RNGT and well for NPRGVS but not for DLAP, while N-10
construct accommodates all three, and are best for DLAP and NPRGVS.
Thus to materialize a native loop of a B-cell epitope, insertion in
both the super-b-stranded, differentially truncated according to
N-10 versus N-5 FG construct in GFP scaffold, maximizes the optimal
outcome.
[0093] Under denaturing conditions, FIG. 7B showed that full length
DE on GFP protein scaffold expressed the linear IgE epitopes.
Products expressed by N-5 versus N-10 on GFP protein scaffold,
exhibited the same intense denatured, linear B-cell epitopes,
despite the deletion of the FG core loop epitope, the residual
super-b-strands and the neighboring amino acids exhibited week
reactivity under denatured conditions with anti-IgE.
[0094] Moreover, deleting the endogenous loop sequences (lane 3 and
lane 4) interfered with the overall folding of FG microdomain,
which also distorted the folding of GFP as shown by loss of
reactivity under even denatured conditions, detected with anti-GFP,
since equal amount of sample of 200 ng were mounted for all
samples. In most instances, reactivities to denatured products did
not have bearing to the native reactivities, since most chemically
conjugated peptides to carrier proteins elicit only anti-peptide
antibodies reactive with linear peptides under denatured
conditions. In contrast, native B-cell core loop B-cell epitopes,
flanked by super b-strands onto GFP protein scaffold, can exhibit
both native conformation-sensitive, native B-cell epitopes under
non-denaturing conditions and linear epitopes under denaturing
conditions. Under these circumstances, the intensity of expression
of C2-3, BC, and DE core loop sequences in the N-5 and N-10
constructs under denatured conditions also correlated with the
relative intensity of native epitopes stoichiometrically.
[0095] GFP, under native conditions, is present as a dimer or
tetramer according to numerous submitted and published X-ray data
to the PDB bank (1GFL). This explained the higher molecular weight
of the FG super-b-strands of different truncations on GFP protein
scaffold exhibited the molecular weight of the tetramer. In
contrast, as shown in Panel A, the various FG and the truncated
constructs exhibited a corresponding 35 KDa band for each species
under denatured conditions (Panel B, FIG. 7B).
[0096] Replacement mutants with the crucial human or mouse IgE
receptor-binding four to six amino acids (i.e., receptor contact
critical residues delineated by X-ray) were performed by
site-specific mutagenesis (SDM). His-purified recombinant products
were assessed by immunoblotting on polyclonal anti-human IgE
(Bethyl), and rabbit and goat anti-murine 26.82 IgE in the lab.
[0097] Summary of Discovery and Embodiment of Super b-Strands
Flanking the Antigenic Loop Sequences
[0098] Through the above numerous designs, recombinant expression,
batch purification and extensive testing according to native and
denaturing conditions, the embodiment of the super-b-strands of as
the minimal, truncated FG microdomain enables vaccine candidates
for all four IgE high affinities receptor-contacting core C2-3, BC,
DE, and FG loops as neutralizing IgE B-cell epitopes. During
initial selection among the four microdomain candidates, we
determine and discover that native b-strands flanking the FG loop
are the most robust b-strands, hence the `super`-b strands,
compared to the other three microdomains that support each of the
respective antigenic core loop sequences. FG microdomain contains
the robust 5' flanking b-strand, followed by HP rigid proline kink,
the native FG core loop sequence, and the robust 3' flanking
b-strand.
[0099] The embodiment of super b-strands for enabling native
expression of native antigenic loops is materialized by the
step-wise experimental discovery. (i) Importantly, the full length
FG microdomain cloned at the N- or C-terminus of GFP enabled
positive albeit weak expression of native FG core loop epitopes,
while other microdomains did not yield native IgE neutralizing
epitopes. (ii) Critically the removal of the N-terminal five amino
acids revealed high antigenicity of the FG loop, and (iii)
truncation of N-terminal ten amino acids of the 5' FG b-strand
secondary structure enabled a stronger antigenic structure,
indicating the importance of the optimal truncation and exhibition
of the flanking super b-strands. (iv) The proline kink immediately
following the 5' b-strands provides additional flanking support for
the core FG loop B-cell epitope, as the truncation of N-terminal 15
amino acids also maintained the native FG loop antigenicity. (v)
Additional primary sequences, C-terminal to the 3' flanking
b-strand flanking are necessary to support the native FG loop,
indicating the essential C-terminal amino acids for the super
b-strands.
[0100] Thus in the embodiment of this invention, the
super-b-strands comprise and are not limited to the N-5, N-10 and
N-15 truncation from the 5' end of FG microdomain, the 5' super
b-stand, proline kink, 3' super b-strand, and the further distal 3'
non-truncated primary sequences. The flanking b-strands of the
truncated FG microdomain serve as the universal scaffolding clamp
for FceRI-contacting core loop sequences of the C2-3L, BC and DE
microdomains, or other B-cell epitopes recombinantly cloned into
the highly thermostable GFP protein scaffold.
[0101] To test long-term safety of the vaccine in rodents, it is
necessary to construct a corresponding set of surrogate rodent
vaccines. Thus the corresponding homologous rodent sequences: C2-3L
(EPRGVI), BC (DLAE) DE (NNATL), and FG (DFPK) loops for this
purpose can be swapped into the human super-b-strands that flank
the native human FG Loop with the native human HLPR sequence
replaced as shown in FIG. 8. The recombinant products can be
detected under native gel running and detection condition by rabbit
anti-26.82 rodent IgE or commercially available neutralizing
anti-rodent IgE.
[0102] Protein Scaffold
[0103] Choice of a thermostable protein scaffold serves as an
embodiment of this invention. GFP is known the most thermostable
protein with T.sub.m at 82.6.degree. C. among all known
calyx-shaped, .beta.-barrel bearing proteins, including lipocalins.
At this temperature, the decimal reduction time is as long as 64
min for quenching 90% of the native fluorescence signal. In
contrast, the melting temperature of lipocalins ranges from 44 to
54.degree. C. with natural phosphotidylethanolamine-binding
lipocalin (PEBP, T.sub.m=54.degree. C.), fluorescein-binding
lipocalin (T.sub.m=44.degree. C.). The engineering step apparently
lowers the Tm by 10.degree. C. via distortion of native
conformation due to insertion or replacement of sequences. Although
the topological similarity of .beta.-can is shared among the
lipocalins and GFP, GFP is far more thermostable than lipocalins by
as much as 38.degree. C. (Skerra, 2000, BBA, 1482:337; Skerra,
2000, J. Mol. Reg. 13: 167). Therefore, GFP poses an advantage in
contrast to lipocalins since the substitution native loops with
random aptameric sequences may render the protein scaffold of GFP
more heat-labile.
[0104] Collectively, the robust protein folding is a prerequisite
for constraining the inserted B-cell loop epitopes in the super
b-strands. Deletion of the native loop sequence without replacement
with other loop sequences can cause collapse of the GFP protein
scaffold with the loss of fluorescence detection. Robust folding of
the protein scaffold plays a critical role in retention of the
native B-cell epitopes of the inserted loops. Thus in the
embodiment of this invention, fluorescence intensity, and positive
immune reactivities to GFP under native conditions serve as
predictor and correlate with the native immunoreactivity of the
swapped B-cell epitopes in the super b-strands recombinantly
expressed onto GFP.
[0105] GFP is also favorably compared to another thermostable
protein scaffold, fibronectin FN3 (T.sub.m=78.degree. C.). The
VEGF-binding, engineered FN3 moieties showed the depressed Tm
ranging from 50 to 65.degree. C., reflecting lower stability by a
magnitude as much as 28.degree. C. due to engineering. Thus the
choice of GFP of a Tm of GFP <82.6.degree. C. may poise as a
more robust protein scaffold in contrast to FN3 in addition to
being a biosensor. In this context, even with a 20.degree. C. drop
in Tm compared to the native GFP, aptameric GFP may still be
favorably compared with single domain, camelid VHH exhibiting a
T.sub.m around 64.degree. C. (Skerra, 2000, BBA, 1482:337; Skerra,
2000, J. Mol. Reg. 13: 167).
[0106] Thus one aspect of the embodiment of the invention resides
in inclusion different protein scaffolds, comprising and are not
limited to GFP, immunoglobulin, camelid VHH, fibronectin, and
lipocalin. Protein scaffolds with different melting temperature in
thermostability can be compared and employed to accommodate the
amino acid sequences of the insert.
[0107] Immunogenicity of FG Loop with Indigenous b-Strands
Integrated and Co-Fold with GFP Protein Scaffold
[0108] Immunogenicity of FG loop with endogenous b-strands
co-folding with GFP: C57BL/6 mice were immunized with 10 .mu.g FG
(N-10)-GFP, and the swapped C2-3L, BC, DE (N-10)-GFP constrained in
the super b-strands of the FG loop on GFP scaffold in alum sc,
boosted twice. Immunoreactivities of antisera with native form of
human IgE were ascertained by its reactivities with the native IgE
at 1,000 to 100,000 fold dilutions by ELISA (plate coated with IgE,
followed by antisera of different dilutions, and rat-anti-mouse
kappa). And the immune sera frequently exhibited (OD reading 2 fold
above background) diluted at 8,000 to 32,000.
[0109] Concomitantly, an IgE neutralizing assay is performed:
Recombinant FceRIa D2/D1 subunit devoid of signal and membrane
anchored sequences of the FceRI holoreceptor
(.alpha..beta..gamma..sub.2) was prepared, expressed with His-tag
and affinity purified via IMAC column. IgE standard can be measured
by IgE capture with his-tagged receptors adsorbed to Ni-treated
96-well plates. FIG. 9A showed IgE BED was captured by the
plate-bound receptors. FIG. 4B showed that sera from FG-10-GFP,
immunized mice abrogated IgE binding to receptors at 1:5,000
dilutions. (cpd, 50 ng/ml control at 1.2 OD), indicating that the
neutralizing antibodies is present at 25 .mu.g/ml, and is capable
of abrogating serum circulating IgE about 1000 IU/ml.
Example 2
FG Loop can be Further Constrained in a Shorter and Redesigned
Cystine Knots (CK) Miniprotein, Min-19 Construct
[0110] Ecballium elaterium trypsin inhibitor II (EETI-II) with 28
amino acids from the squash family was the first discovered CK knot
miniprotein (37). EETI-II has a triple anti-parallel .beta.-sheet
of consisting of three b-strands, knotted within with three cystine
disulfide knots (forming the respective cysteine 1/4, 2/5, and 3/6
pair) (Gracy, 2008, N. A. R., 36: suppl 1:314). The folding
requirement for all three cystine pairs to form in the oxidized
environment poses stringent conditions for the native triple
stranded b-sheets to additionally constrain/conform the molecularly
grafted foreign B-cell epitopes or those flanked by the super
b-strands.
[0111] An embodiment of this invention is to reduce the complexity
by eliminating one cystine bridge. An evolutionarily conserved
cystine knot motif, distributed throughout mammalian proteins,
includes only two disulfide bridges constraining the .beta.-hairpin
loop structures. Min-23, deleted of one cystine pair, exhibits a
well-defined conformation, similar to the structure of the native
parent inhibitor EETI II in folding. Min-23 is thermostable, folded
with the cystine bridges supported by the C2-C5 (residue 9/21) and
the C3-C6 (residue 15/27) (Heitz, 1999, Biochem, 38: 10615).
[0112] The integrity of endogenous triple b-stranded, b-sheets of
Min-23 is deemed to play a constraining role on the peptide
conformation of the loop. Loop 1, 2, 3 and Loop 5 can be considered
for replacement of IgE B-cell epitopes, and loop 5 in
cystine-knotted peptides is endowed with longer amino acid
sequences, and can be considered for accommodating longer peptide
sequences.
[0113] Insertion of foreign sequence directly between the native
loop 5' sequence has been performed between proline (P) and
asparagine (N) (Souriau, 2005, Biochem, 44: 7143). The direct
insertion of foreign sequences with native undeleted sequences
combined can lead to distortion of the inserted sequences without
the necessary beta-sheet in the grafted sequence. On the other
hand, the insertion of foreign sequences into the complete loop 5
deleted sequences can also affect the formation of the necessary
beta sheet of the inserted sequences.
[0114] To strengthen the constraining the capacity of loop 5,
Min-23 is modified into Min-19 or Min-18: An embodiment of this
invention resides in preserving the b-sheet structure of the
foreign inserted sequence in the modified deleted loop 5 with
retained hydrophobic phenylalanine, while the inserted structure
also possess its own super b-strands. Min-19 and Min-18 are
equipped with the indigenous and acquired b-strands supported by
the double cystine bridges that offer the additional rigidity and
constraining capacity for inserted foreign peptides with
cystine-stabilized b-sheet (CSB).
[0115] In the overall embodiment of this invention, we choose to
insert the endogenous b-strands scaffolded FG loop with the
accompanying super b-strands into the C-FC (Cys---PheCys) position
of Min-23 without perturbing the indigenous b-strands in the stable
two disulfide bonded cystine knots.
[0116] Min-23 retains also high thermal stability, with a mean
T.sub.m of 100.degree. C., folded with the cystine bridges
supported by the C2-C5 (residues 9/21) and the C3-C6 (residues
15/27). The molecular construct proceeds as follows: (i)
Site-directed mutagenesis (SDM) was conducted to eliminate the
first five residues of trypsin inhibitor sequences, including the
first cysteine (at position 2) from the EETI-II-GFP construct (16,
18), while maintaining the first b-strand starting at residue Met
(at position 7). (ii) The subsequent SDMs were performed to render
cysteine 19.fwdarw.serine 19 in order to deplete the cystine
1-->4 bridge, and also to retain the residue 21, phenylalanine
(F). (iii) Foreign loop sequences were inserted by SDM by primer
extension.
[0117] FIG. 10 showed the sequence of regular Min-23, and the
Min-23 onto C-FC construct, and also the Min-19 construct with F
left in loop 5 (CC) on GFP and the containment of truncated FG in
single, duplicate and triplet repeats. FIG. 11 showed that HindIII
site and the inclusive sequences of GFP vector, and the insertion
of the FG microdomain in the Min-19.
[0118] As shown in FIG. 11, various truncated FG loops with the
flanking b-strands, as well as cancatemers: FG (-10Nx2; FG-15Nx3)
were inserted between C-FC of Min-19, ranging from 19 to 57 amino
acids, and these constructs with the terminal residues, FC of
Min-23 were separated by (gly)3 spacer (the 5' glycine also can be
regarded as the last and natural amino acid sequence from Min-23,
hence the construct of Min-19) onto the Hind III site of the GFP at
the N-terminus with its His-tag added at the C-terminus of the GFP
protein scaffold.
[0119] As shown in FIG. 12, these constructs were expressed
following IPTG induction, and purified by the IMAC column. FIG. 12
showed not only a single FG can be constrained in Min-23 (lane 2,
3, 5 for FG-5, FG-10, and FG-15) but also a duplicate (bidentate,
FG-15, lane 6), and a triplicate (tridentate, FG-10, lane 4) FG
loops with variously trimmed flanking b-strands, can be
accommodated with the Min-23 cystine knots, detected under native
conditions by pAb anti-IgE as .about.120 KDa tetramer. In contrast,
the construct with the deleted FG core loop sequence was not
detected by pAb (lane 1).
[0120] In contrast, FIG. 13A showed diverse varieties of
homo-bidentate versions, and hetero-bidentate, i.e., bifunctional
versions of truncated FG and C2-3 microdomains in Min-19. Although
FG N-5 single version of Min-19 preserved the native
immunoreactivity (lane 7), the homo-bidentate lacked native immune
reactivity, indicating a distortion of the native conformation via
its duplicate presence of FG-N-5 in the Min-19 (lane 1). Since
truncated C2-3 did not exhibit the native determinant (lane 12, 13
for the single and lane 4 for bidentate), the immune reactivity of
bifunctional of C2-3 18AA, 3' and FG N-5 is due to the reactivity
with FG-N-5, and thus affirming the loss of reactivity in the
bidentate FG-N-5 was likely due to crowding and the distortion of
native B-cell epitope.
[0121] Therefore, the retention of native immune reactivities of
homo-bidentate FG-N-10 (lane 3), and FG-N-15 (lane 11) and the
homo-tridentate FG-N-10 (lane 9) indicated the robustness and the
advantage of shorten version of the FG microdomain with regard to
their insertion into the Min-19 in GFP protein scaffold.
[0122] In contrast to the robust FG loop presentation in the Min-19
construct, different versions of C2-3 exhibited weak expression of
native immune reactivity to anti-neutralizing IgE as shown in FIG.
13, i.e., C2-3 18 from 3' bidentate (lane 4), or bifunctional from
N- or C-terminal to FG N-5 (lane 2 and lane 5) and as single 23
from 3' (lane 12), and 31 from 3' (lane 13).
[0123] This detailed mapping indicates that (i) Min-19 version can
cause presentation of IgE loop epitope already properly scaffolded
by the super b-strands of FG microdomain, and (ii) the Min-19
accommodation leads to only moderate expression of the B-cell
epitopes, C2-3 with no intrinsic scaffolding secondary structures;
(iii) the retention of C2-3 core loop B-cell epitope can be
materialized in swapping with the native FG loop core sequence in
the super-b-strands of FG microdomain in Min-19, supported by the
observation in FIG. 7A.
[0124] FIG. 13 B summaries the observation in the histogram, which
also indicates the critically important supporting role of the 3'
amino acid sequence for the consolidating the super b-strands. The
deletion of the seven amino acids strongly diminished the capacity
of FG microdomain to preserve the native FG loop B-cell
epitopes.
[0125] Therefore, the embodiment of the invention resides in
integrating the super b-strands into loop 5 properly selected for
endogenous loop deletion. It is important to note that M-19 can
consistently maintain the conformation of the three variety of
trimmed FG super b-stranded loop epitope to the same intensity of
expression. Thus Min-19 GFP construct at the N-terminal of GFP
reproduced the same pattern of conformation constraint without
compromising the native epitope scaffolded by the preexisting
scaffold.
[0126] Importance of Pre-Existing Secondary Structure Constraint
for the Loop Sequences
[0127] The inventive process consists of empirically testing the
feasibility of various configurations of EETI-II and Min-23 series
in constraining loop B-cell epitope. Several modalities loop 5
insertion are included in the embodiment of this invention: (i)
depending on the nature of B-cell epitope and the extent of
truncation of the super b-strands, entire replacement of the native
loop 5 with the new B-cell epitope may be implemented; (ii) the
B-cell epitope can be inserted following the rigid proline (P) with
N and G deleted, and neighboring the P, in utilizing the proline
kink as a pivot for the foreign epitope.
[0128] (iii) The B-cell epitope can be inserted immediately prior
to the hydrophobic phenylanaline residue in the truncated loop 5.
The feasibility of each conformation relies on the nature of amino
acid composition of the foreign sequences to be inserted. (iv)
Min-23 was employed for accommodating sequences between P and N
(18) without the definition of secondary structures.
[0129] (v) Direct loop to loop swapping in that the B-cell loop
sequence (without the flanking secondary structure) may be swapped
with the indigenous sequence in the native loop of Min-19; (vi) in
addition to accommodating the B-cell epitope in N-terminal end
folding with GFP protein produced in the bacterial cytosol, folding
and production in the oxidative periplasmic space constitutes
another embodiment of this invention.
[0130] In the modality direct loop to loop swapping, and
recombinant proteins expressed the presence of oxidative folding
environment (v and vi), we herein demonstrated the critical role of
super b-strands for the core IgE neutralizing epitopes: BC (VDLAPS)
and DE (QRNGTL) loops as shown in loop swap into the super b-stands
of FG microdomain (FIGS. 6 and 7). FIG. 14 showed the design and
execution diagram of the direct loop swap between BC and DE loop
sequences with loop sequences of the loop 1, 2, 3, 5 of the EETI-II
in pMal that can be produced in the favorable oxidative periplasmic
space.
[0131] Next, we evaluate whether the series of insertion mutants
were recombined at the C-terminus of the maltose-binding protein
(MBP) vector, and expressed in the more oxidative periplasmic
microenvironment may improve the folding, and restore the native
loop conformation of the grafted BC and DE core loop epitopes.
[0132] The yield of MBP protein was elevated; however as shown in
FIG. 15, EETI-II with inserted BC and DE loops were weakly reactive
with neutralizing anti-IgE under native conditions. Therefore, the
embodiment of this invention attests to the sequence-dependent,
direct loop to loop swap, i.e., native loop 1, 2, 3, 5 of the
EETI-II with foreign loop sequences, and further indicates the
requirement of loop sequences integrated into super b-strands prior
to its replacement of the native loop of EETI-II
[0133] The lack of direct loop swapping between the BC and DE loops
and the native loop sequences of the EETI-II, substantiates the
need for the main embodiment of the invention in (i) employing a
truncated EETI-II with two cystine bridges to reduce the errors of
knots formation of the existing cysteines; (ii) scaffolding the
foreign loops preferably in super b-strands when further
constrained in the double cystine knots. Hence, the embodiment of
the invention resides in the two-step process of scaffolding the
loop sequence within preexisting super b-strands, and of
integrating the constrained loop into the optimized Min-23
series.
[0134] Enablement of Strongly Augmented Expression of Super
b-Stranded IgE B-Cell Epitope in Min-18 in pMbp in Oxidative
Periplasmic Space
[0135] Next we determine the role the oxidative microenvironment in
augmenting yield and intensity of the native epitope pre-scaffolded
by super b-strands in the Min-18 construct in pMal in the optimal
oxidative folding milieu. Min-18 with various N- and C-terminal FG
deletions were presented in FIG. 16 executed Diagram and Min-18
with different lengths of C2-3 is presented in FIG. 17 executed
Diagram.
[0136] As shown in FIG. 18, Min-23 did serve this further
augmentation for the FG epitope which was already stabilized by the
super b-strands, and the IgE B-cell epitopes were strongly
augmented in the oxidative periplasmic milieu, as observed in lane
2 of FG with 5 amino acid deletion at the N-terminus, and loss the
reactivity for 15 amino acid deletion and 18 amino acid deletion
from the N-terminus. The intensity of expression of the equivalent
concentrations of maltose column affinity purified product exceeds
that of even 150 ng of human IgE, exhibiting all IgE B-cell
epitopes, including also four receptor-docking IgE B-cell
epitope.
[0137] Further the FG segment with as few as three or five amino
acid deletion from the C-terminus, 3, 5, 10 (lane 5, 6, and 7)
materialized a detrimental effect on the native conformation of the
FG epitopes. This further confirms that integrity of C-terminus of
FG microdomain is indispensable as noted in also the
FG-microdomain-pGFP construct (cpd. FIG. 13B).
[0138] As shown in FIG. 19, noticeably, in addition to the FG N-5,
Min-18 plays a robust role in further constraining and augmenting
expression of the FG N-10 pMal construct in the periplasmic
oxidative environment. The intensity of expression of even the
recombinant products from the crude bacterial product also far
exceeded that of even 200 ng of human IgE, which exhibited all IgE
B-cell epitopes, including four receptor-docking IgE B-cell
epitopes and other non-receptor-related B-cell epitopes from CHe1
to CHe4 domains. Thus the B-cell monospecific vaccine candidate
dictates that a native single epitope in the embodiment of the
invention be equivalent to or exceeds that of high concentrations
of native molecules.
[0139] Hence, the steps materialize IgE B-cell N-minus ten amino
acid truncated FG epitope can be a good IgE vaccine candidate. Thus
the summary statement of one aspect of the embodiment of this
invention is that intense robust expression of a super b-stranded
scaffolded epitope can be augmented by Min-23 series, Min-18 as a
second step of cystine-knotted scaffolding/framing and three
dimensional protein folding, consummated in the Min23-pMal
oxidative chemical folding milieu.
[0140] As shown in FIG. 20, the further constraint exerted by the
double cystine bridges can enhance the scaffolding of even the weak
secondary structure for the C-terminal 22 amino acids of the C2-3
microdomain. As shown in FIG. 20, denatured and native
conformation, the effect of constraining C2-3 with cystine knots is
effective.
[0141] One main embodiment of this invention was described as
swapping the B-cell loop epitopes with native FG loop sequences of
the super b-strands of the FG microdomain (FIGS. 6 and 7).
Therefore in another embodiment of this invention, IgE B-cell
vaccines can be materialized in a two-step process into Min-18 in
pMal for augmented expression. In step 1, C2-3 core loop epitope,
or BC core loop epitope, or DE core loop epitope can be swapped and
replace the native loop sequences of super b-strands of the
truncated FG microdomain. And in step 2, the antigenic loops
scaffolded by the super b-strands can be further constrained by
Min-19, expressed onto the protein scaffold that permits expression
and oxidative folding.
[0142] In addition to IgE B-cell epitopes, another embodiment of
this invention includes swapping the viral neutralizing epitopes of
gp41, gp120 of HIV and hemagglutinin of influenza virus into the
super b-strands of the truncated FG microdomain. Thus the
embodiment of this invention for preparing general neutralizing
vaccines for HIV, Flu virus are: (i) the discrete loop structure
for viral amino acids delineated through its attachment to the host
receptors will be cloned into the flanking super b-strands; (ii)
the viral neutralizing epitope in the flanking super b-strands is
inserted into the Min-23 loop 5 fused with pMal; (iii) the viral
epitope within super b-strands pMal construct is expressed in the
oxidative periplasmic space folding environment.
[0143] In summary, the embodiment of the invention for enabling
specific IgE vaccine application is an integration of the four
steps: (i) the step of validating the super b-strands is taken for
conducting truncation of the N- and C-terminal amino acids; this
leads to the conclusion of critical N (N-5, and N-10) and
C-terminus (C-0) in flanking the FG loop regions; (ii) the
insertion of other critical IgE receptor contacting amino acid
residues will be constructed, substituting the native FG loop;
(iii) the Min-23 loop 5 insertion further strengthens and
stabilizes the super b-strands constructs; (iv) the oxidative
folding environment of MBP in the periplasmic space will provide
the folding environment.
Example 3
Methods and Protocols for Super b-Strands, Min-23 and GFP Scaffold
Constructs
[0144] Reagents employed are: Phusion.RTM. Flash High-Fidelity PCR
Master Mix, restriction enzymes, and pMAL-p5E vector were purchased
from New England Biolabs. Antibodies were from Abcam (goat anti-GFP
antibodies, goat anti-human IgE antibodies, and HRP conjugated
donkey polyclonal to Goat IgG), Clontech (Full-length GFP
polyclonal antibodies), and Cell Signaling Technology (HRP
conjugated anti-mouse IgG antibodies and HRP-conjugated anti-rabbit
IgE antibodies). TMB Membrane Peroxidase Substrate System was
ordered from KPL. Rapid DNA ligation kit was from Roche. DNA
purification kits were purchased from QIAGEN. Ready Gels were from
Bio-Rad. Immobilon-P Transfer Membrane was from Millipore. Vector
pGFPuv was from Clontech.
[0145] PCR reaction: DNA template (1-10 ng) and primers (0.25
.mu.M) are added to distilled water to a final volume of 25 .mu.l.
Equal volume of Phusion Flash PCR Master Mix is added and mixed.
The PCR conditions are: denature at 98.degree. C. for 1 second,
annealing at 55.degree. C. for 5 seconds, and extension at
72.degree. C. for 15 second/1 kb. Run 25-30 cycles. After the
cycles, the samples are extended for another 1 minute, and hold at
4.degree. C.
[0146] Expression of recombinant proteins in E. coli: Selective
colonies of IgE-GFP constructs were picked up and inoculated in 1
ml LB medium with appropriate antibiotics (ampicillin,
chloramphenicol, or spectinomycin at 100 .mu.g/ml, 25 .mu.g/ml and
80 .mu.g/ml, respectively). The cultures were grown at 37.degree.
C. for overnight. Next day, add 3 ml fresh LB medium with inducer
(Isopropyl .beta.-D-1-thiogalactopyranoside (IPTG) to 1 mM or
chlorotetracycline (CTC) to 100 ng/ml) to the overnight culture,
and grew the culture for another 1-4 hours to induce protein
expression. Cells were harvested and washed with 1.times.PBS twice.
Cell walls were degraded with lysozyme (1 mg/ml) in 1.times.PBS
buffer for 15 minutes at room temperature. Then, the cells were
sonicated on ice for three rounds, 10 seconds each at 50% power
with 30 seconds intervals. After spin at 12,000 rpm for 10 minutes,
the supernatant was transferred to a new tube for further
analysis.
[0147] Protein electrophoresis, native or denaturing conditions:
For native gel electrophoresis, cell lysates were mixed with equal
volume 2.times. native sample buffer (0.125 M Tris-HCl, 5%
glycerol, pH6.8) just prior to loading samples onto native gel.
Proteins were separated with native running buffer (3.03 g Tris
Base, 14.4 g Glycine, in 1000 ml distilled water). For denaturing
conditions, samples were mixed with sodium dodecyl sulfate (SDS)
Reducing buffer (final SDS and .beta.-mercaptoethanol
concentrations are 1%), and heated at 95.degree. C. for 5 minutes
before loading onto gels. The running buffer contains 1% SDS. After
separation, proteins were transferred to PVDF membranes for
immunoblotting.
[0148] Immunoblotting assay. PVDF membrane with transferred
proteins was blocked with 5% dry milk in PBS for 1 hour. After
washing three times with PBST (1.times.PBS with 0.05% Tween-20),
the membrane was incubated with primary antibodies (1:1000 to
1:10,000 dilution according to vendor's instructions) for 1-16
hours. Wash the membrane three times with PBST and incubate with
HRP conjugated second antibodies for 1 hour. After washing the
membrane three times with PBST, TMB Membrane Peroxidase Substrate
(enhancer: TMB peroxidase substrate: peroxidase substrate solution
B=1:5:5) was added to cover the membrane. To stop the
colorimetrical reaction, distilled water was added at the desired
color.
[0149] Generation of EETI-II-IgE (8AA) Constructs
[0150] As EETI-II cDNA is only 84 nucleotides (28 amino acids), the
EETI-II-IgE (8AA, "DSNPRGVS") constructs were generated by PCR of
two synthesized primers. Restriction enzyme site (Hind III) and
linker (between EETI-II and GFPuv) were added in the primers.
TABLE-US-00001 The primers for pEETI-L1-8AA (replacing EETI-II loop
1 with 8 C2-3 linker residues): EETI-L1-8AA-F:
5'cgccaagcttggggtgcgattccaacccgagaggggtgagctgcaaacaggactccgactgcctggctggc--
3' EETI-L1-8AA-R:
5'-tcataagcttcggatctcttaatccgcagaaaccgttgggcccgcaaacgcagccagccaggcagtcggag-
-3' The primers for pEETI-L2-8AA (replacing EETI-II loop 2 with 8
C2-3 linker residues): EETI-L2-8AA-F:
5'-cgccaagcttggggtgcccgcgaatcctaatgcgttgcgattccaacccgagaggggtgagctgcctggct-
gg-3' EETI-L2-8AA-R:
5'-tcataagcttcggatctcttaatccgcagaaaccgttgggcccgcaaacgcagccagccaggcagctcacc-
c-3' The primers for pEETI-L3-8AA (replacing EETI-II loop 3 with 8
C2-3 linker residues): EETI-L3-8AA-F:
5'-cgccaagcttggggtgcccgcgaatcctaatgcgttgcaaacaggactccgactgcgattccaacccgaga-
gggg-3' EETI-L3-8AA-R:
5'-tcataagcttcggatctcttaatccgcagaaaccgttgggcccgcaaacgcagctcacccctctcgggttg-
gaatc-3' The primers for pEETI-L5-8AA (replacing EETI-II loop 5
with 8 C2-3 linker residues): EETI-L5-8AA-F:
5'-cgccaagcttggggtgcccgcgaatcctaatgcgttgcaaacaggactccgactgcctggctggctgcgtt-
tg-3' EETI-L5-8AA-R:
5'-tcataagcttcggatctcttaatccgcagctcacccctctcgggttggaatcgcaaacgcagccagccagg-
cag-3'. Primers used to substitute EETI-II loop 1: EETI1 add QRNGTL
sense: caagcttggggtgcCAAAGAAACGGTACTCTTtgcaaacaggactc EETI1 add
QRNGTL antisense: gagtcctgtttgcaAAGAGTACCGTTTCTTTGgcaccccaagcttg
EETI1 add VDLAPS sense:
caagcttggggtgcGTTGATCTTGCTCCATCTtgcaaacaggactc EETI1 add VDLAPS
antisense: gagtcctgtttgcaAGATGGAGCAAGATCAACgcaccccaagcttg Primers
used to substitute EETI-II loop 2: EETI-L2 add QRNGTL sense:
cctaatgcgttgcCAAAGAAACGGTACTCTTtgcctggctggctg EETI-L2 add QRNGTL
antisense: cagccagccaggcaAAGAGTACCGTTTCTTTGgcaacgcattagg EETI-L2
add VDLAPS sense: cctaatgcgttgcGTTGATCTTGCTCCATCTtgcctggctggctg
EETI-L2 add VDLAPS antisense:
cagccagccaggcaAGATGGAGCAAGATCAACgcaacgcattagg Primers used to
substitute EETI-II loop 3: EETI-L3 add QRNGTL sense:
ggactccgactgcCAAAGAAACGGTACTCTTtgcgtttgcgggc EETI-L3 add QRNGTL
antisense: gcccgcaaacgcaAAGAGTACCGTTTCTTTGgcagtcggagtcc EETI-L3 add
VDLAPS sense: ggactccgactgcGTTGATCTTGCTCCATCTtgcgtttgcgggc EETI-L3
add VDLAPS antisense: gcccgcaaacgcaAGATGGAGCAAGATCAACgcagtcggagtcc
Primers used to substitute EETI-II loop 5: EETI-L5 add QRNGTL
sense: ctggctgcgtttgcCAAAGAAACGGTACTCTTtgcggaggaggacc EETI-L5 add
QRNGTL antisense: ggtcctcctccgcaAAGAGTACCGTTTCTTTGgcaaacgcagccag
EETI-L5 add VDLAPS sense:
ctggctgcgtttgcGTTGATCTTGCTCCATCTtgcggaggaggacc EETI-L5 add VDLAPS
antisense: ggtcctcctccgcaAGATGGAGCAAGATCAACgcaaacgcagccag
[0151] Site-Directed Mutagenesis (SDM):
Nucleotide can be substituted, added or deleted by site-directed
mutagenesis. Synthesis primers contain the modified nucleotide(s).
1 .mu.l of the forward primer at 125 ng/.mu.l, and 1 .mu.l of
reverse primer at 125 ng/.mu.l to 1 ul template at 50 ng/.mu.l to
25 .mu.l PCR master mix and 22 .mu.l dd water in a total of 50
.mu.l reaction. Take reaction and cycle as follows: 1) 98.degree.
C. for 10 sec; 2) 98.degree. C. for 5 sec and 68.degree. C. for 1
minute 15 sec/kb vector, repeat 18 cycles. After the cycles, the
samples are extended at 68.degree. C. for 10 min, and hold at
4.degree. C. Dpn I (1 .mu.l) is added to each PCR reaction, and
incubated at 37.degree. C. for one hour. Take 1 .mu.l for
transformation.
[0152] Construct of EETI-IgE peptide (QRNGTL, and VDLAPS) into loop
1, 2, 3, 5 and fused to c-terminus of MBP: PCR products from EET1
in GFPuv-His were used as template with modification by adding Not1
(5') and EcoR1 (3') ends with removal of Gly-Gly linker, and
ligated into c-terminus of pMal5pE.
1) This forward primer (EETI-WT) works for all empty cassettes and
all substitutions EXCEPT loop 1 delete and loop 1 substitutions:
GATCgcggccgc (Not1)GGGtgc (L1 cys) CCGCGAATCCTA. 2) This reverse
primer works for all empty cassettes and all substitutions EXCEPT
loop 5 delete and loop 5 substitutions: GATCgaattc (EcoR1)tccgca
(L5 cys) GAAACCGTTGGG. 3) This forward primer GATCgcggccgc
(Not1)GGGtgc (L1 cys) (QRNG), will produce a product consisting of
an EET1, Loop 1 substitute QRNGTL with Not1/EcoR1 ends. 4) This
forward primer will amplify the product from the GFPuv-His EET1
Loop 1 substitute VDLAPS: GATCgcggccgc (Not1)GGGtgc (L1 cys)
(VDLA). 5) This forward primer EETI: GATCgcggccgc (Not1)GGGtgctgc
(L1 deletion)AAACAGGAC, will produce a product consisting of EET1
wt with a Loop 1 deletion and Not1/EcoR1 ends (Loop 1 EET1 empty
cassette). 6) This reverse primer: GATCgaattc (EcoR1)tccgca (L5
cys)AAGAGTACCGTT, will produce a product consisting of EET1 with a
Loop 5 substitution of QRNGTL with Not1/EcoR1 ends. 7) This reverse
primer GATCgaattc (EcoR1)tccgca (L5cys) AGATGGAGCA, will produce a
product consisting of EET1 with a Loop 5 substitution of VDLAPS
with Not1'EcoR1 ends. 8) This reverse primer GATCgaattc
(EcoR1)tccgcagca (L5 deletion)AACGCAGCC, will produce a product
consisting of EET1 with a Loop 5 deletion and Not1/EcoR1 ends.
[0153] Ligation reactions are as follow:
a). Primers 1 and 2+templates as follows: GFPuv-His wt; GFPuv-His
deletion Loop 2; GFPuv-His deletion Loop 3; GFPuv-His substitution
Loop 2 QRNGTL; GFPuv-His substitution Loop 2 VDLAPS; GFPuv-His
substitution Loop 3 QRNGTL; GFPuv-His substitution Loop 3 VDLAPS.
b). Primers 2 and 5+template GFPuv-His Loop 1 deletion. c). Primers
2 and 3+template GFPuv-His Loop 1 substitution of QRNGTL. d)
Primers 2 and 4+template GFPuv-His Loop 1 substitution of VDLAPS.
e) Primers 1 and 8+template GFPuv-His Loop 5 deletion. f) Primers 1
and 6+template GFPuv-His Loop 5 substitution of QRNGTL g) Primers 1
and 7+template GFPuv-His Loop 5 substitution of VDLAPS. Final
constructs from the ligation reactions are: 1). pMal-p5e+EET1 wt;
2). pMal-p5e+EET1 del L1; 3). pMal-p5e+EET1 del L2; 4).
pMal-p5e+EET1 del L3; 5). pMal-p5e+EET1 del L5; 6). pMal-p5e+EET1
sub QRNGTL Loop 1; 7). pMal-p5e+EET1 sub QRNGTL Loop 2; 8).
pMal-p5e+EET1 sub QRNGTL Loop 3; 9). pMal-p5e+EET1 sub QRNGTL Loop
5; 10). pMal-p5e+EET1 sub VDLAPS Loop 1; 11). pMal-p5e+EET1 sub
VDLAPS Loop 2; 12). pMal-p5e+EET1 sub VDLAPS Loop 3; 13).
pMal-p5e+EET1 sub VDLAPS Loop 5
[0154] Construct of Min-23
Min23 is constructed by PCR reaction with template: GFPuv-His EET1
wt with deleted loop 5, and forward primer: GATCgcggccgc
(Not1)TTGCAAACAGGAC; and the reverse primer: GATCgaattc
(EcoR1)TCCgcagca (delete loop 5) AACGCAGCCAGCC. The PCR fragment
was then digested with Not1 and EcoR1 and ligated into c terminus
of pMalp5E vector cut with Not1/EcoR1 with the following
sequence:
TABLE-US-00002 CTAATGCGTTGCAAACAGGACTCCGACTGCCTGGCTGGCTGCG TT
tgctgcGGA
[0155] Construct of MBP fused Min-23 (with deleted loop 5) or
Min-18 construct: EETI-GFPuv-His with deleted loop 5 was used as
template for PCR. The two primers are: forward primer-Not I
(5'-GATCgcggccgcCTAATGCGTTGCAAACAGGAC-3') and reverse primer-EcoRI
(5'-GATCgaattcTCCgcagcaAACGCAGCCAGCC-3'). The PCR product was
digested with NotI and EcoRI and cloned into pMAL-p5E vector
between NotI and EcoRI sites. MBP and EETI-II will be expressed as
a fusion protein.
[0156] Construct of 16 amino acids of FG IgE peptide addition to
loop 5 of Min23 with three constituents: Overall strategy is to use
forward and reverse primer in a site directed mutagenesis reaction
with pMal-p5e EET1-Min23 delete Loop 5 as a template. Thus Min23
template with deleted loop 5 and with the Not1 and EcoR1 at the two
flanking sites is as follows: Gcggccgc
(Not1)CTAATGCGTTGCAAACAGGACTCCGACTGCCTGGCTGGCT GCGTTtgc (with loop
5 deleted, between two cys tgc) tgcGGAgaattc (EcoR1).
Forward 23 min 16AA SDM Primer:
ggctgcgtttgcGATTCCAACCCGAGAGGGGTGAGCGCCTACCTAAGCCGG CCCAGCCCG
tgcGGAgaattc, which is designed according to the three
constituents: (i) 5' flanking region to EETI Loop 5: ggctgcgtttgc;
(ii) 16 AA IgE domain insert into L-5:
TABLE-US-00003 D S N P R G V S A Y L S GAT TCC AAC CCG AGA GGG GTG
AGC GCC TAC CTA AGC R P S P C CGG CCC AGC CCG; (iii) tgcGGAgaattc
(EcoR1).
Reverse 23 min 16AA SDM primer is as follows:
gaattctccgcaCGGGCTGGGCCGGCTTAGGTAGGCGCTCACCCCTCTCGGGTT
GGAATCgcaAACGCAGCC, designed according to three constituents (i)
EcoR1 3' most EET1, Gaattctccgca (cysteine); (ii) 16 AA IgE domain
(Rev) insert into L-5:
TABLE-US-00004 P S P R S L Y A S V G R CGG GCT GGG CCG GCT TAG GTA
GGC GCT CAC CCC TCT P N S D C CGG GTT GGA ATC; (iii) gca (cysteine)
AACGCAGCC
[0157] Construct of truncated FG in pGFP or Min-23-pGFP:
Alternatively, forward and reverse primers with HindIII at the 5'
and 3' with end with various triplet deletions were added to
mini-IgE pGFP, and micro-IgE domain (pFG-GFPuv) for PCR reactions,
and the products were digested with HindIII and ligated with
HindIII digested GFPuv with C-terminal six histamine for IMAC
column purification. To have further secondary cystine bridge
constraint built in, N-5, N-10, and N-15 oligos Forward primers are
designed with starting 5' deletion or N-- deletion of five, ten or
fifteen triplets (N-5, N-10, N-15), or with reverse primers of 3'
deletion or C-deletion of 7 triplets (C-7). To place the various
truncated FG versions into Min-19 cystine knot, site-directed
mutagenesis forward primers with 5' flanking region to EETI Loop 5:
ggctgcgtttgc, followed by triplets of N-5, N-10, and N-15 were
added to Min-23-pGFP, and reverse primer, starting gca (cysteine)
AACGCAGCC.
[0158] To prepare bidentate or tridentate structure of the
truncated FG/Min-23-pGFP, PCR fragments were prepared from the 5'
to 3' HindIII sites of the first generation of monomeric truncated
FG-Min 23-pGFP construct, the PCR fragment was purified and
digested with HindIII and ligated to the first generation of
monomeric truncated FG-Min 23-GFP vectors. DNA sequences were
performed and the length of the concatemer evaluated, and bidentate
or tridentate configuration was then determined.
[0159] Construct Mini- and Micro-IgE: The mini-IgE fragments were
amplified from human IgE heave chain cDNA constructed in the
laboratory. The primers used for PCR are: IgE C2-3-F (Hind III)
(5'-GATCAAGCTTGcgcacctacacctgccaggtc-3') and IgE FG-loop-R (AgeI)
(5'-GATCACCGGTACacgcgggccgctggtcttgg-3'). PCR product was digested
with Hind III and AgeI and cloned into pGFPuv between Hind III and
AgeI. The micro-IgE constructs were generated by site-directed
mutagenesis. The primers used were:
TABLE-US-00005 Delete BC, DE, and FG loops (pC2-3-GFPuv):
del(BCDEFG): 5'-ccggcccagcccggtaccggtagaaa-3';
del(BCDEFG)-antisense: 5'-tttctaccggtaccgggctgggccgg-3'; Delete
C2-3 linker (pBCDEFG-GFPuv): del(C2-3 linker):
5'-tgattacgccaagcttgttcgacctgttcatccg-3'; del(C2-3
linker)-antisense: 5'-cggatgaacaggtcgaacaagcttggcgtaatca-3'; Delete
C2-3 linker and BC loop (pDEFG-GFPuv): del(C2-3 + BC):
5'-gattacgccaagcttggtgaaccactccacca-3'; del(C2-3 + BC)-antisense:
5'-tggtggagtggttcaccaagcttggcgtaatc-3' Delete C2-3 linker, BC loop
and DE loop (pFG-GFPuv): del(C2-3BCDE):
5'-gattacgccaagcttgacccgagactggatcg-3'; del(C2-3BCDE)-antisense:
5'-cgatccagtctcgggtcaagcttggcgtaatc-3'.
Example 4
Illustrative Word Diagram
[0160] Part I:
SDM/Primer Extension for Deletion of EETI Cystine Knot Loops 1, 2,
3, and 5
[0161] Take pGFPuv-HIS EETI wt (N-terminal) construct and make
primers for site directed mutagenesis (SDM, primer extension):
[0162] 1. Loop 1 deletion using site directed mutagenesis:
TABLE-US-00006 End Lac Start HindIII EET1 WT loop 1
ATGACCATGATTACGCCAAGCTTGGGGtgcCCGCGAATCCTAATGCGTtgc loop 2 loop 3
loop 5 AAACAGGACTCCGACtgcCTGGCTGGCtgcGTTtgcGGGCCCAACGGTTTCtgc Gly
Gly HindIII GFPuv GGAGGAGGACCAAGCTTGATGAGTAAAGGAGAA Delete Loop 1
of EETI End Lac Start HindIII EETI C delete Loop 1 C
ATGACCATGATTACGCCAAGCTTGGGGtgcCCGCGAATCCTAATGCGTtgcAAACAGGACTCC
GACTGCCTGGCTGGCTGCGTTTGCGGGCCCAACGGTTTCTG Gly Gly HindIII GFPuv
CGGAGGAGGACCAAGCTTGATGAGTAAAGGAGAA Primers to delete Loop 1: C C
Del-loop1-sense 5'-ccaagcttggggtgctgcaaacaggactcc-3'
Del-loop1-antisense 5'-ggagtcctgtttgcagcaccccaagcttgg-3'
[0163] 2. Loop 2 deletion using site directed mutagenesis:
TABLE-US-00007 Delete Loop 2 End Lac Start HindIII EETI WT
ATGACCATGATTACGCCAAGCTTGGGGTGCCCGCGAATCCTAATGCGT Delete Loop 2
tgcAAACAGGACTCCGACtgcCTGGCTGGCTGCGTTTGCGGGCCCAACG Gly Gly HindIII
GFPuv GTTTCTGCGGAGGAGGACCAAGCTTGATGAGTAAAGGAGAA Primers to delete
Loop 2: C C Del-loop2-sense 5'-atcctaatgcgttgctgcctggctgg ctgc-3'
Del-loop2-antisense 5'-gcagccagccaggcagcaacgcatta ggat-3'
[0164] 3. Loop 3 deletion using site directed mutagenesis:
TABLE-US-00008 Delete loop 3 End Lac Start HindIII EET1 WT
ATGACCATGATTACGCCAAGCTTGGGGTGCCCGCGAATCCTAATGCGTT Delete Loop 3
GCAAACAGGACTCCGACtgcCTGGCTGGCtgcGTTTGCGGGCCCAACGG Gly Gly HindIII
GFPuv TTTCTGCGGAGGAGGACCAAGCTTGATGAGTAAAGGAGAA Primers to delete
Loop 3: C C Del-loop3-sense 5'-ggactccgactgctgcgtttgcggg c-3'
Del-loop3-antisense 5'-gcccgcaaacgcagcagtcggagtc c-3'
[0165] 4. Loop 5 deletion using site directed mutagenesis:
TABLE-US-00009 Delete Loop 5 of EETI End Lac Start HindIII EET1 WT
ATGACCATGATTACGCCAAGCTTGGGGTGCCCGCGAATCCTAATGCGTT Delete
GCAAACAGGACTCCGACTGCCTGGCTGGCTGCGTTtgcGGGCCCAACG Loop 5 Gly Gly
HindIII GFPuv GTTTCtgcGGAGGAGGACCAAGCTTGATGAGTAAAGGAGAA Primers to
delete Loop 5: C C Del-loop5-sense 5'-ggctggctgcgtttgctgcggaggag
gaccaag-3' Del-loop5-antisense 5'-cttggtcctcctccgcagcaaacgca
gccagcc-3'
[0166] Part II:
[0167] SDM/Primer Extension for Addition of IgE Core Loop C2-3. BC,
DE Sequences into EETI Cystine Knot Loops 1, 2, 3, and 5 Deleted
Mutants [0168] 1. Add QRNGTL and VDLAPS to GFPuv-His EETI (N term)
Loop 1 deletion using site directed mutagenesis:
TABLE-US-00010 [0168] Primers needed to add QRNGTL substitution in
loop 1: ETTI L1 add QRNGTL sense caagcttggggtgc tgcaaacaggactc ETTI
L1 add QRNGTL antisense gagtcctgtttgca gcaccccaagcttg deleted End
Lac CCGCGAAT Start HindIII EETI add: Q R ATGACCATGATTACG
CCAAGCTTGGGGtgc Loop 1 CCTAATGCGT N G T L tgcAAACAGGACTC
CGACTGCCTGGCTGGCTGCGTTTG Gly Gly HindIII GFPuv
CGGGCCCAACGGTTTCTGCGGAGGAGGACCAAGCTTGATGAGTAAAGGAG AA Primers
needed to add VDLAPS substitution in loop 1: ETTI L1 add VDLAPS
sense caagcttggggtgc tgcaaacaggactc ETTI L1 add VDLAPS antisense
gagtcctgtttgca gcaccccaagcttg deleted End Lac CCGCGAAT Start
HindIII EETI add: V D L ATGACCATGATTACGC CAAGCTTGGGGtgc Loop 1
CCTAATGCGT A P S tgcAAACAGGACTC CGACTGCCTGGCTGGCTGCGTTTG Gly Gly
HindIII GFPuv CGGGCCCAACGGTTTCTGCGGAGGAGGACCAAGCTTGATGAGTAAAGGA
GAA
[0169] 2. Add QRNGTL and VDLAPS to GFPuv-His (c term) construct
with EETI Loop 2 deletion using site directed mutagenesis:
TABLE-US-00011 [0169] Primers needed to add QRNGTL substitution in
loop 2: ETTI-L2 add QRNGTL sense cctaatgcgttgc tgcctggctggctg
ETTI-L2 add QRNGTL antisense cagccagccaggca gcaacgcattagg End Lac
Start HindIII EETI WT ATGACCATGATTACGCCAAGCTTGGGGTGCCCGCGAAT
CCTAATGCGT Delete Loop 2 AAACAGGACTCCGAC add: Q R N G T L tgc
tgcCTGGCTGGCTG CGTTTGCGG GCCCAACGGTTTCTG Gly Gly HindIII GFPuv
CGGAGGAGGACCAAGCTTGATGAGTAAAGGAGAA Primers needed to add VDLAPS
substitution in loop 2: ETTI-L2 add VDLAPS sense acctaatgcgttgc
tgcctggctggctg ETTI-L2 add VDLAPS antisense cagccagccaggca
gcaacgcattagg End Lac Start HindIII EETI WT
ATGACCATGATTACGCCAAGCTTGGGGTGCCCGCGAAT CCTAATGCGT Delete Loop 2
AAACAGGACTCCGAC add: V D L A P S tgc tgcCTGGCTGGCTG CGTTTGCGG
GCCCAACGGTTTCTG Gly Gly HindIII GFPuv
CGGAGGAGGACCAAGCTTGATGAGTAAAGGAGAA
[0170] 3. Add QRNGTL and VDLAPS to GFPuv-His construct with EETI (N
term) Loop 3 deletion using site directed mutagenesis:
TABLE-US-00012 [0170] End Lac Start HindIII EET1 WT
ATGACCATGATTACGCCAAGCTTGGGGTGCCCGCGAATCCTAATGCGT Delete Loop 3
CTGGCTGGC add: Q R N G T L TGCAAACAGGACTCCGACtgc tgcGTTT Gly Gly
HindIII GFPuv GCGGGCCCAACGGTTTCTGCGGAGGAGGACCAAGCTTGATGAGTAAAGG
AGAA Primers needed to add QRNGTL substitution in loop 3: ETTI-L3
add QRNGTL sense ggactccgactgc tgcgtttgcgggc ETTI-L3 add QRNGTL
antisense gcccgcaaacgca gcagtcggagtcc End Lac Start HindIII EET1 WT
ATGACCATGATTACGCCAAGCTTGGGGTGCCCGCGAATCCTAATGCGTT Delete Loop 3
CTGGCTGGC add: V D L A P S GCAAACAGGACTCCGACtgc tgcGTTTG Gly Gly
HindIII GFPuv CGGGCCCAACGGTTTCTG CGGAGGAGGACCAAGCTTGATGAGTAAAGGA
GAA Primers needed to add VDLAPS substitution in loop 3: ETTI-L3
add VDLAPS sense ggactccgactgc tgcgtttgcgggc ETTI-L3 add VDLAPS
antisense gcccgcaaacgca gcagtcggagtcc
[0171] 4. Add QRNGTL and VDLAPS to GFPuv-His construct with EETI (N
term) Loop 5 deletion using site directed mutagenesis:
TABLE-US-00013 [0171] End Lac Start HindIII EET1 WT
ATGACCATGATTACGCCAAGCTTGGGGTGCCCGCGAATCCTAATGCGTTG Delete GGGCCC
add: Q R N CAAACAGGACTCCGACTGCCTGGCTGGCTGCGTTtgc Loop 5 AACGGTTTC G
T L Gly Gly HindIII GFPuv tgcGGAGGAGGACCAAGCTTGATGAGTAAAGGAGAA
Primers needed to add QRNGTL substitution in loop 5: ETTI-L5 add
QRNGTL sense ctggctgcgtttgcCAAAGAAACGGTACTCTTtgcggaggaggacc ETTI-L5
add QRNGTL antisense ggtcctcctccgcaAAGAGTACCGTTTCTTTGgcaaacgcagccag
End Lac Start HindIII EET1 WT
ATGACCATGATTACGCCAAGCTTGGGGTGCCCGCGAATCCTAATGCGTT Delete GGGCCC
add: V D L GCAAACAGGACTCCGACTGCCTGGCTGGCTGCGTTtgc Loop 5 AACGGTTTC
A P S Gly Gly HindIII GFPuv tgcGGAGGAGGACCAAGCTTGATGAGTAAAGGAGAA
Primers needed to add VDLAPS substitution in loop 5: ETTI-L5 add
VDLAPS sense ctggctgcgtttgcGTTGATCTTGCTCCATCTtgcggaggaggacc ETTI-L5
add VDLAPS antisense
ggtcctcctccgcaAGATGGAGCAAGATCAACgcaaacgcagccag
[0172] PCR conditions for the above reactions: 25 ul 2.times.
Phusion flash master mix; 1 ul forward primer of a 1:4 dilution of
100 uM solution; 1 ul reverse primer of a 1:4 dilution of 100 uM
solution; 1 ul template of a 100 ng/ul solution; 22 ul ddH.sub.2O
in a total of 50 ul. The cycling conditions are: 1) 98.degree. C.
for 10 sec; 2) 98.degree. C. for 1 sec, 55.degree. C. for 15 sec,
72.degree. C. for 15 sec, and repeat 31 cycles; 3) 72.degree. C.
for 1 min. PCR products were cleaned with QiaQuick PCR purification
kit, digested, and run on 2% agarose gel, and bands were cut out
band and purified with QiaQuick gel extraction kit cut vectors were
ligated with the PCR product with rapid DNA ligation kit from
Roche, and transform 2 ul of reaction into 50 ul DH5 competent
cells and plate on Amp LB plates.
Site-directed mutagenesis for loop deletion conditions: Phusion
Flash (NEB) 25 ul PCR master mix; 1 ul forward primer at 125 ng/ul;
1 ul Reverse primer at 125 ng/ul; 1 ul template at 50 ng/ul. 25 ul
PCR master mix; 22 ul ddH.sub.2O in a total 50 ul. Take reaction
and cycle as follows: 1) 98.degree. C. for 10 sec; 2) 98.degree. C.
for 5 sec and 68.degree. C. for 1 minute at the rate of 15 sec/kb
vector for a total 18 cycles; 3) 68.degree. C. for 10 min
[0173] Part III:
1). Min-23 with deleted loop 5, i.e., Min-18 construct; 2). Delete
loop 5 except P, i.e., Min-19 construct; 3). Min-23 construct on
pMal:
[0174] 1) and 2): For Min-18 and Min 19 constructs
TABLE-US-00014 Sequence to be obtained:
CTAATGCGTTGCAAACAGGACTCCGACTGCCTGGCTGGCTGCGTT tgc(C) tgc (C)GGA, or
tgc(C) TTC(P) tgc(C) GGA With the Forward primer: GATC (Not
1)CTAATGCGTTGCAAACAGGAC; Reverse primer: GATC (EcoRI)TCCgcagca
(C-C, deleted loop 5) AACGCAGCCAGCC. Alternatively: GATC
(EcoRI)TCCgcaTTCgca (C-C, deleted loop 5 except TTC, phenylanaline)
AACGCAGCCAGCC.
For the PCR reaction, use the forward and reverse primer and
GFPuv-His EET1 wt with the above deleted loop 5 EETI wild type as
the template. Digest PCR fragments with Not1 and EcoR1 and ligate
into c terminus of pMalp5E vector cut with Not1/EcoR1.
3). For Min-23:
TABLE-US-00015 [0175] (i) Wild type and truncated sequences: wt
EET1 start of Min-23
gggtgcccgcgaatcCTAATGCGTTGCAAACAGGACTCCGACTGCCTGG
CTGGCTGCGTTTGCGGGCCCAACGGTTTCTGCGGA 10 20 30 40 50 gggtgcccgcgaatc
(EETI 5' deleted) cccacgggcgcttag (G C P R I) (Min-23 start)
CTAATGCGTTGCAAACAGGACTCCGACTGCCTGGCTGGCTGCGTTTGCGG
GATTACGCAACGTTTGTCCTGAGGCTGACGGACCGACCGACGCAAACGCC L M R C K Q D S
D C L A G C V C G 60 GCCCAACGGTTTCTGCGGA CGGGTTGCCAAAGACGCCT P N G
F C G (ii) Primer design: Min23 For PCR primer with Not1 ends: Not1
GATC CTAATGCGTTGCAAACAGGAC forward primer Min23 Rev PCR primer with
EcoR1 ends: GATC (EcoRI)TCCGCAGAAACCGTTGGGCCC reverse primer
[0176] Procedures for constructs: Forward and reverse primers in
were added in a PCR reaction with GFPuv-His EET1 wt as the
template: Gcggccgc (Not 1) (Min-23:
CTAATGCGTTGCAAACAGGACTCCGACTGCCTGGCTGGCTGCGTTTGCGGGCCCA
ACGGTTTCTGCGGAgaattc (EcoRI). PCR product was digested with
Not1/EcoR1, and ligated to pMAL 5pE that was digested with Not1 and
EcoR1 on the C terminus of the maltose gene with the removal of the
Gly-Gly-linker from pMal, and the fused PCR fragment was then
cloned into Not1/EcoR1 pMal5pE.
Sequence CWU 1
1
5211920DNAhomo sapienshuman IgE 1ggatccctgc cacggggtcc ccagctcccc
catccaggcc ccccaggctg atgggcgctg 60gcctgaggct ggcactgact aggttctgtc
ctcacagcct ccacacagag cccatccgtc 120ttccccttga cccgctgctg
caaaaacatt ccctccaatg ccacctccgt gactctgggc 180tgcctggcca
cgggctactt cccggagccg gtgatggtga cctgggacac aggctccctc
240aacgggacaa ctatgacctt accagccacc accctcacgc tctctggtca
ctatgccacc 300atcagcttgc tgaccgtctc gggtgcgtgg gccaagcaga
tgttcacctg ccgtgtggca 360cacactccat cgtccacaga ctgggtcgac
aacaaaacct tcagcggtaa gagagggcca 420agctcagaga ccacagttcc
caggagtgcc aggctgaggg ctggcagagt gggcaggggt 480tgagggggtg
ggtgggctca aacgtgggaa cacccagcat gcctggggac ccgggccagg
540acgtgggggc aagaggaggg cacacagagc tcagagaggc caacaaccct
catgaccacc 600agctctcccc cagtctgctc cagggacttc accccgccca
ccgtgaagat cttacagtcg 660tcctgcgacg gcggcgggca cttccccccg
accatccagc tcctgtgcct cgtctctggg 720tacaccccag ggactatcaa
catcacctgg ctggaggacg ggcaggtcat ggacgtggac 780ttgtccaccg
cctctaccac gcaggagggt gagctggcct ccacacaaag cgagctcacc
840ctcagccaga agcactggct gtcagaccgc acctacacct gccaggtcac
ctatcaaggt 900cacacctttg aggacagcac caagaagtgt gcaggtacgt
tcccacctgc cctggtggcc 960gccacggagg ccagagaaga ggggcgggtg
ggcctcacac agccctccgg tgtaccacag 1020attccaaccc gagaggggtg
agcgcctacc taagccggcc cagcccgttc gacctgttca 1080tccgcaagtc
gcccacgatc acctgtctgg tggtggacct ggcacccagc aaggggaccg
1140tgaacctgac ctggtcccgg gccagtggga agcctgtgaa ccactccacc
agaaaggagg 1200agaagcagcg caatggcacg ttaaccgtca cgtccaccct
gccggtgggc acccgagact 1260ggatcgaggg ggagacctac cagtgcaggg
tgacccaccc ccacctgccc agggccctca 1320tgcggtccac gaccaagacc
agcggtgagc catgggcagg ccggggtcgt gggggaaggg 1380agggagcgag
tgagcggggc ccgggctgac cccacgtctg gccacaggcc cgcgtgctgc
1440cccggaagtc tatgcgtttg cgacgccgga gtggccgggg agccgggaca
agcgcaccct 1500cgcctgcctg atccagaact tcatgcctga ggacatctcg
gtgcagtggc tgcacaacga 1560ggtgcagctc ccggacgccc ggcacagcac
gacgcagccc cgcaagacca agggctccgg 1620cttcttcgtc ttcagccgcc
tggaggtgac cagggccgaa tgggagcaga aagatgagtt 1680catctgccgt
gcagtccatg aggcagcgag cccctcacag accgtccagc gagcggtgtc
1740tgtaaatccc ggtaaatgac gtactcctgc ctccctccct cccagggctc
catccagctg 1800tgcagtgggg aggactggcc agaccttctg tccactgttg
caatgacccc aggaagctac 1860ccccaataaa ctgtgcctgc tcagagcccc
agtacaccca ttcttgggag cgggcagggc 19202574PRThomo sapienshuman IgE
2Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val 1
5 10 15 His Ser Gln Thr Gln Leu Val Gln Ser Gly Ala Glu Val Arg Lys
Pro 20 25 30 Gly Ala Ser Val Arg Val Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Ile 35 40 45 Asp Ser Tyr Ile His Trp Ile Arg Gln Ala Pro
Gly His Gly Leu Glu 50 55 60 Trp Val Gly Trp Ile Asn Pro Asn Ser
Gly Gly Thr Asn Tyr Ala Pro 65 70 75 80 Arg Phe Gln Gly Arg Val Thr
Met Thr Arg Asp Ala Ser Phe Ser Thr 85 90 95 Ala Tyr Met Asp Leu
Arg Ser Leu Arg Ser Asp Asp Ser Ala Val Phe 100 105 110 Tyr Cys Ala
Lys Ser Asp Pro Phe Trp Ser Asp Tyr Tyr Asn Phe Asp 115 120 125 Tyr
Ser Tyr Thr Leu Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val 130 135
140 Ser Ser Ala Ser Thr Gln Ser Pro Ser Val Phe Pro Leu Thr Arg Cys
145 150 155 160 Cys Lys Asn Ile Pro Ser Asn Ala Thr Ser Val Thr Leu
Gly Cys Leu 165 170 175 Ala Thr Gly Tyr Phe Pro Glu Pro Val Met Val
Thr Trp Asp Thr Gly 180 185 190 Ser Leu Asn Gly Thr Thr Met Thr Leu
Pro Ala Thr Thr Leu Thr Leu 195 200 205 Ser Gly His Tyr Ala Thr Ile
Ser Leu Leu Thr Val Ser Gly Ala Trp 210 215 220 Ala Lys Gln Met Phe
Thr Cys Arg Val Ala His Thr Pro Ser Ser Thr 225 230 235 240 Asp Trp
Val Asp Asn Lys Thr Phe Ser Val Cys Ser Arg Asp Phe Thr 245 250 255
Pro Pro Thr Val Lys Ile Leu Gln Ser Ser Cys Asp Gly Gly Gly His 260
265 270 Phe Pro Pro Thr Ile Gln Leu Leu Cys Leu Val Ser Gly Tyr Thr
Pro 275 280 285 Gly Thr Ile Asn Ile Thr Trp Leu Glu Asp Gly Gln Val
Met Asp Val 290 295 300 Asp Leu Ser Thr Ala Ser Thr Thr Gln Glu Gly
Glu Leu Ala Ser Thr 305 310 315 320 Gln Ser Glu Leu Thr Leu Ser Gln
Lys His Trp Leu Ser Asp Arg Thr 325 330 335 Tyr Thr Cys Gln Val Thr
Tyr Gln Gly His Thr Phe Glu Asp Ser Thr 340 345 350 Lys Lys Cys Ala
Asp Ser Asn Pro Arg Gly Val Ser Ala Tyr Leu Ser 355 360 365 Arg Pro
Ser Pro Phe Asp Leu Phe Ile Arg Lys Ser Pro Thr Ile Thr 370 375 380
Cys Leu Val Val Asp Leu Ala Pro Ser Lys Gly Thr Val Asn Leu Thr 385
390 395 400 Trp Ser Arg Ala Ser Gly Lys Pro Val Asn His Ser Thr Arg
Lys Glu 405 410 415 Glu Lys Gln Arg Asn Gly Thr Leu Thr Val Thr Ser
Thr Leu Pro Val 420 425 430 Gly Thr Arg Asp Trp Ile Glu Gly Glu Thr
Tyr Gln Cys Arg Val Thr 435 440 445 His Pro His Leu Pro Arg Ala Leu
Met Arg Ser Thr Thr Lys Thr Ser 450 455 460 Gly Pro Arg Ala Ala Pro
Glu Val Tyr Ala Phe Ala Thr Pro Glu Trp 465 470 475 480 Pro Gly Ser
Arg Asp Lys Arg Thr Leu Ala Cys Leu Ile Gln Asn Phe 485 490 495 Met
Pro Glu Asp Ile Ser Val Gln Trp Leu His Asn Glu Val Gln Leu 500 505
510 Pro Asp Ala Arg His Ser Thr Thr Gln Pro Arg Lys Thr Lys Gly Ser
515 520 525 Gly Phe Phe Val Phe Ser Arg Leu Glu Val Thr Arg Ala Glu
Trp Glu 530 535 540 Gln Lys Asp Glu Phe Ile Cys Arg Ala Val His Glu
Ala Ala Ser Pro 545 550 555 560 Ser Gln Thr Val Gln Arg Ala Val Ser
Val Asn Pro Gly Lys 565 570 3108DNAhomo sapiensC2-3 DNA 3cgcacctata
cctgccaggt gacctatcag ggccatacct ttgaagatag caccaaaaaa 60tgcgcggata
gcaacccgcg cggcgtgagc gcgtatctga gccgcccg 108436PRTHomo sapiensC2-3
peptide 4Arg Thr Tyr Thr Cys Gln Val Thr Tyr Gln Gly His Thr Phe
Glu Asp 1 5 10 15 Ser Thr Lys Lys Cys Ala Asp Ser Asn Pro Arg Gly
Val Ser Ala Tyr 20 25 30 Leu Ser Arg Pro 35 590DNAHomo sapiensBC
loop 5agcccgtttg atctgtttat tcgcaaaagc ccgaccatta cctgcctggt
ggtggatctg 60gcgccgagca aaggcaccgt gaacctgacc 90630PRTHomo
sapiensBC loop peptide 6Ser Pro Phe Asp Leu Phe Ile Arg Lys Ser Pro
Thr Ile Thr Cys Leu 1 5 10 15 Val Val Asp Leu Ala Pro Ser Lys Gly
Thr Val Asn Leu Thr 20 25 30 781DNAHomo sapiensDE loop 7aaaccggtga
accatagcac ccgcaaagaa gaaaaacagc gcaacggcac cctgaccgtg 60accagcaccc
tgccggtggg c 81827PRTHomo sapiensDE loop peptide 8Lys Pro Val Asn
His Ser Thr Arg Lys Glu Glu Lys Gln Arg Asn Gly 1 5 10 15 Thr Leu
Thr Val Thr Ser Thr Leu Pro Val Gly 20 25 9102DNAHomo sapiensFG
loop 9acccgcgatt ggattgaagg cgaaacctat cagtgccgcg tgacccatcc
gcatctgccg 60cgcgcgctga tgcgcagcac caccaaaacc agcggcccgc gc
1021034PRTHomo sapiensFG loop peptide 10Thr Arg Asp Trp Ile Glu Gly
Glu Thr Tyr Gln Cys Arg Val Thr His 1 5 10 15 Pro His Leu Pro Arg
Ala Leu Met Arg Ser Thr Thr Lys Thr Ser Gly 20 25 30 Pro Arg
1187DNAHomo sapiensFG loop N-5 11gagggcgaga cctaccagtg cagggtgacc
cacccccacc tgcccagggc cctgatgagg 60agcaccacca agaccagcgg ccccagg
871229PRTHomo sapiensFG loop N-5 peptide 12Glu Gly Glu Thr Tyr Gln
Cys Arg Val Thr His Pro His Leu Pro Arg 1 5 10 15 Ala Leu Met Arg
Ser Thr Thr Lys Thr Ser Gly Pro Arg 20 25 1372DNAHomo sapiensFG
loop N-10 13cagtgcaggg tgacccaccc ccacctgccc agggccctga tgaggagcac
caccaagacc 60agcggcccca gg 721424PRTHomo sapiensFG loop N-10
peptide 14Gln Cys Arg Val Thr His Pro His Leu Pro Arg Ala Leu Met
Arg Ser 1 5 10 15 Thr Thr Lys Thr Ser Gly Pro Arg 20 1557DNAHomo
sapiensFG loop 15 15cacccccacc tgcccagggc cctgatgagg agcaccacca
agaccagcgg ccccagg 571619PRTHomo sapiensFG loop N-15 peptide 16His
Pro His Leu Pro Arg Ala Leu Met Arg Ser Thr Thr Lys Thr Ser 1 5 10
15 Gly Pro Arg 1775DNAHomo sapiensFG N-10 del HLPR 17gagggcgaga
cctaccagtg cagggtgacc caccccgccc tgatgaggag caccaccaag 60accagcggcc
ccagg 751825PRTHomo sapiensFG N-10 del HLPR peptide 18Glu Gly Glu
Thr Tyr Gln Cys Arg Val Thr His Pro Ala Leu Met Arg 1 5 10 15 Ser
Thr Thr Lys Thr Ser Gly Pro Arg 20 25 1945DNAHomo sapiensFG N-15
del HLPR 19caccccgccc tgatgaggag caccaccaag accagcggcc ccagg
452015PRTHomo sapiensFG N-15 de; HLPR peptide 20His Pro Ala Leu Met
Arg Ser Thr Thr Lys Thr Ser Gly Pro Arg 1 5 10 15 2187DNAHomo
sapiensFG N-5 del HLPR 21gagggcgaga cctaccagtg cagggtgacc
cacccccacc tgcccagggc cctgatgagg 60agcaccacca agaccagcgg ccccagg
872212DNAHomo sapiensHLPR 22cacctgccca gg 12234PRTHomo sapiensHLPR
peptide 23His Leu Pro Arg 1 2427DNAHomo sapiensmin half super-beta
24caccccacca agaccagcgg ccccagg 27259PRTHomo sapiensmin half
super-beta peptide 25His Pro Thr Lys Thr Ser Gly Pro Arg 1 5
2675DNAHomo sapienstwo super beta with tail 26tatcagtgcc gcgtgaccca
tccgcatctg ccgcgcgcgc tgatgcgcag caccaccaaa 60accagcggcc cgcgc
752725PRTHomo sapienstwo super-beta with tail peptide 27Tyr Gln Cys
Arg Val Thr His Pro His Leu Pro Arg Ala Leu Met Arg 1 5 10 15 Ser
Thr Thr Lys Thr Ser Gly Pro Arg 20 25 2854DNAHomo sapienstwo
super-beta Min 28tatcagtgcc gcgtgaccca tccgcatctg ccgcgcgcgc
tgatgcgcag cacc 542918PRTHomo sapienstwo super-beta Min peptide
29Tyr Gln Cys Arg Val Thr His Pro His Leu Pro Arg Ala Leu Met Arg 1
5 10 15 Ser Thr 3036DNAHomo sapiensHP with right beta 30catccgcatc
tgccgcgcgc gctgatgcgc agcacc 363112PRTHomo sapiensHP with right
beta peptide 31His Pro His Leu Pro Arg Ala Leu Met Arg Ser Thr 1 5
10 3224DNAHomo sapiensHP del HLPR with right beta 32catccggcgc
tgatgcgcag cacc 243342DNAHomo sapienstwo beta plus HP 33tatcagtgcc
gcgtgaccca tccggcgctg atgcgcagca cc 423414PRTHomo sapienstwo beta
plus HP peptide 34Tyr Gln Cys Arg Val Thr His Pro Ala Leu Met Arg
Ser Thr 1 5 10 3536DNAHomo sapienstwo beta 35tatcagtgcc gcgtgaccgc
gctgatgcgc agcacc 363612PRTHomo sapienstwo beta peptide 36Tyr Gln
Cys Arg Val Thr Ala Leu Met Arg Ser Thr 1 5 10 3718DNAHomo
sapiensone beta left 37tatcagtgcc gcgtgacc 18386PRTHomo sapiensone
beta left peptide 38Tyr Gln Cys Arg Val Thr 1 5 3918DNAHomo
sapiensone beta right 39gcgctgatgc gcagcacc 18406PRTHomo sapiensone
beta right 40Ala Leu Met Arg Ser Thr 1 5 4154DNAEcballium
elateriumMin-18 41ctaatgcgtt gcaaacagga ctccgactgc ctggctggct
gcgtttgctg cgga 544218PRTEcballium elateriumMin-18 peptide 42Leu
Met Arg Cys Lys Gln Asp Ser Asp Ser Leu Ala Gly Cys Val Cys 1 5 10
15 Cys Gly 4357DNAEcballium elateriumMin-19 43ctaatgcgtt gcaaacagga
ctccgactgc ctggctggct gcgtttgctt ctgcgga 574419PRTEcballium
elateriumMin-19 peptide 44Leu Met Arg Cys Lys Gln Asp Ser Asp Ser
Leu Ala Gly Cys Val Cys 1 5 10 15 Phe Cys Gly 4569DNAEcballium
elateriumMin-23 45ctaatgcgtt gcaaacagga ctccgactgc ctggctggct
gcgtttgcgg gcccaacggt 60ttctgcgga 694623PRTEcballium
elateriumMin-23 peptide 46Leu Met Arg Cys Lys Gln Asp Ser Asp Cys
Leu Ala Gly Ser Val Cys 1 5 10 15 Gly Pro Asn Gly Phe Cys Gly 20
4784DNAEcballium elateriumEETI-II 47gggtgcccgc gaatcctaat
gcgttgcaaa caggactccg actgcctggc tggctgcgtt 60tgcgggccca acggtttctg
cgga 844828PRTEcballium elateriumEETI-II peptide 48Gly Cys Pro Arg
Ile Leu Met Arg Cys Lys Gln Asp Ser Asp Cys Leu 1 5 10 15 Ala Gly
Cys Val Cys Gly Pro Asn Gly Phe Cys Gly 20 25 491161DNAEscherichia
coliMBP 49atgaaaattg aagaaggcaa actggtgatt tggattaacg gcgataaagg
ctataacggc 60ctggcggaag tgggcaaaaa atttgaaaaa gataccggca ttaaagtgac
cgtggaacat 120ccggataaac tggaagaaaa atttccgcag gtggcggcga
ccggcgatgg cccggatatt 180attttttggg cgcatgatcg ctttggcggc
tatgcgcaga gcggcctgct ggcggaaatt 240accccggata aagcgtttca
ggataaactg tatccgttta cctgggatgc ggtgcgctat 300aacggcaaac
tgattgcgta tccgattgcg gtggaagcgc tgagcctgat ttataacaaa
360gatctgctgc cgaacccgcc gaaaacctgg gaagaaattc cggcgctgga
taaagaactg 420aaagcgaaag gcaaaagcgc gctgatgttt aacctgcagg
aaccgtattt tacctggccg 480ctgattgcgg cggatggcgg ctatgcgttt
aaatatgaaa acggcaaata tgatattaaa 540gatgtgggcg tggataacgc
gggcgcgaaa gcgggcctga cctttctggt ggatctgatt 600aaaaacaaac
atatgaacgc ggataccgat tatagcattg cggaagcggc gtttaacaaa
660ggcgaaaccg cgatgaccat taacggcccg tgggcgtgga gcaacattga
taccagcaaa 720gtgaactatg gcgtgaccgt gctgccgacc tttaaaggcc
agccgagcaa accgtttgtg 780ggcgtgctga gcgcgggcat taacgcggcg
agcccgaaca aagaactggc gaaagaattt 840ctggaaaact atctgctgac
cgatgaaggc ctggaagcgg tgaacaaaga taaaccgctg 900ggcgcggtgg
cgctgaaaag ctatgaagaa gaactggcga aagatccgcg cattgcggcg
960accatggaaa acgcgcagaa aggcgaaatt atgccgaaca ttccgcagat
gagcgcgttt 1020tggtatgcgg tgcgcaccgc ggtgattaac gcggcgagcg
gccgccagac cgtggatgaa 1080gcgctgaaag atgcgcagac caacagcagc
agcaacaaca acaacaacaa caacaacaac 1140aacctgggca ttgaaggccg c
116150387PRTEscherichia coliMBP protein 50Met Lys Ile Glu Glu Gly
Lys Leu Val Ile Trp Ile Asn Gly Asp Lys 1 5 10 15 Gly Tyr Asn Gly
Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr 20 25 30 Gly Ile
Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe 35 40 45
Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala 50
55 60 His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu
Ile 65 70 75 80 Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe
Thr Trp Asp 85 90 95 Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr
Pro Ile Ala Val Glu 100 105 110 Ala Leu Ser Leu Ile Tyr Asn Lys Asp
Leu Leu Pro Asn Pro Pro Lys 115 120 125 Thr Trp Glu Glu Ile Pro Ala
Leu Asp Lys Glu Leu Lys Ala Lys Gly 130 135 140 Lys Ser Ala Leu Met
Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro 145 150 155 160 Leu Ile
Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys 165 170 175
Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly 180
185 190 Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala
Asp 195 200 205 Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly
Glu Thr Ala 210 215 220 Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn
Ile Asp Thr Ser Lys 225 230 235 240 Val Asn Tyr Gly Val Thr Val Leu
Pro Thr Phe Lys Gly Gln Pro Ser 245
250 255 Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser
Pro 260 265 270 Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu
Leu Thr Asp 275 280 285 Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro
Leu Gly Ala Val Ala 290 295 300 Leu Lys Ser Tyr Glu Glu Glu Leu Ala
Lys Asp Pro Arg Ile Ala Ala 305 310 315 320 Thr Met Glu Asn Ala Gln
Lys Gly Glu Ile Met Pro Asn Ile Pro Gln 325 330 335 Met Ser Ala Phe
Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala 340 345 350 Ser Gly
Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr Asn 355 360 365
Ser Ser Ser Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Leu Gly Ile 370
375 380 Glu Gly Arg 385 51720DNAAequorea victoriaGFP 51atgagtaaag
gagaagaact tttcactgga gttgtcccaa ttcttgttga attagatggt 60gatgttaatg
ggcacaaatt ttctgtcagt ggagagggtg aaggtgatgc aacatacgga
120aaacttaccc ttaaatttat ttgcactact ggaaaactac ctgttccatg
gccaacactt 180gtcactactt tctcttatgg tgttcaatgc ttttcccgtt
atccggatca tatgaaacgg 240catgactttt tcaagagtgc catgcccgaa
ggttatgtac aggaacgcac tatatctttc 300aaagatgacg ggaactacaa
gacgcgtgct gaagtcaagt ttgaaggtga tacccttgtt 360aatcgtatcg
agttaaaagg tattgatttt aaagaagatg gaaacattct cggacacaaa
420ctcgagtaca actataactc acacaatgta tacatcacgg cagacaaaca
aaagaatgga 480atcaaagcta acttcaaaat tcgccacaac attgaagatg
gatccgttca actagcagac 540cattatcaac aaaatactcc aattggcgat
ggccctgtcc ttttaccaga caaccattac 600ctgtcgacac aatctgccct
ttcgaaagat cccaacgaaa agcgtgacca catggtcctt 660cttgagtttg
taactgctgc tgggattaca catggcatgg atgagctcta caaataatga
72052238PRTAequorea victoriaGFP protein 52Met Ser Lys Gly Glu Glu
Leu Phe Thr Gly Val Val Pro Ile Leu Val 1 5 10 15 Glu Leu Asp Gly
Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu 20 25 30 Gly Glu
Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys 35 40 45
Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe 50
55 60 Ser Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys
Arg 65 70 75 80 His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val
Gln Glu Arg 85 90 95 Thr Ile Ser Phe Lys Asp Asp Gly Asn Tyr Lys
Thr Arg Ala Glu Val 100 105 110 Lys Phe Glu Gly Asp Thr Leu Val Asn
Arg Ile Glu Leu Lys Gly Ile 115 120 125 Asp Phe Lys Glu Asp Gly Asn
Ile Leu Gly His Lys Leu Glu Tyr Asn 130 135 140 Tyr Asn Ser His Asn
Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn Gly 145 150 155 160 Ile Lys
Ala Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val 165 170 175
Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 180
185 190 Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu
Ser 195 200 205 Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu
Glu Phe Val 210 215 220 Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu
Leu Tyr Lys 225 230 235
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